Novel compounds

ABSTRACT

The invention provides BASB231 polypeptides and polynucleotides encoding BASB231 polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are diagnostic, prophylactic and therapeutic uses.

FIELD OF THE INVENTION

This invention relates to polynucleotides, (herein referred to as“BASB231 polynucleotide(s)”), polypeptides encoded by them (referred toherein as “BASB231” or “BASB231 polypeptide(s)”), recombinant materialsand methods for their production. In another aspect, the inventionrelates to methods for using such polypeptides and polynucleotides,including vaccines against bacterial infections. In a further aspect,the invention relates to diagnostic assays for detecting infection ofcertain pathogens.

BACKGROUND OF THE INVENTION

Haemophilus influenzae is a non-motile Gram negative bacterium. Man isits only natural host.

H. influenzae isolates are usually classified according to theirpolysaccharide capsule. Six different capsular types designated athrough f have been identified. Isolates that fail to agglutinate withantisera raised against one of these six serotypes are classified as nontypeable, and do not express a capsule.

The H. influenzae type b is clearly different from the other types inthat it is a major cause of bacterial meningitis and systemic diseases.non typeable H. influenzae (NTHi) are only occasionally isolated fromthe blood of patients with systemic disease.

NTHi is a common cause of pneumonia, exacerbation of chronic bronchitis,sinusitis and otitis media.

Otitis media is an important childhood disease both by the number ofcases and its potential sequelae. More than 3.5 millions cases arerecorded every year in the United States, and it is estimated that 80%of children have experienced at least one episode of otitis beforereaching the age of 3 (1). Left untreated, or becoming chronic, thisdisease may lead to hearing loss that can be temporary (in the case offluid accumulation in the middle ear) or permanent (if the auditivenerve is damaged). In infants, such hearing losses may be responsiblefor delayed speech learning.

Three bacterial species are primarily isolated from the middle ear ofchildren with otitis media: Streptococcus pneumoniae, NTHi and M.catarrhalis. These are present in 60 to 90% of cases. A review of recentstudies shows that S. pneumoniae and NTHi each represent about 30%, andM. catarrhalis about 15% of otitis media cases (2). Other bacteria canbe isolated from the middle ear (H. influenzae type B, S. pyogenes, . .. ) but at a much lower frequency (2% of the cases or less).

Epidemiological data indicate that, for the pathogens found in themiddle ear, the colonization of the upper respiratory tract is anabsolute prerequisite for the development of an otitis; other factorsare however also required to lead to the disease (3-9). These areimportant to trigger the migration of the bacteria into the middle earvia the Eustachian tubes, followed by the initiation of an inflammatoryprocess. These other factors are unknown todate. It has been postulatedthat a transient anomaly of the immune system following a viralinfection, for example, could cause an inability to control thecolonization of the respiratory tract (5). An alternative explanation isthat the exposure to environmental factors allows a more importantcolonization of some children, who subsequently become susceptible tothe development of otitis media because of the sustained presence ofmiddle ear pathogens (2).

Various proteins of H. influenzae have been shown to be involved inpathogenesis or have been shown to confer protection upon vaccination inanimal models.

Adherence of NTHi to human nasopharygeal epithelial cells has beenreported (10). Apart from fimbriae and pili (11-15), many adhesins havebeen identified in NTHi. Among them, two surface exposedhigh-molecular-weight proteins designated HMW1 and HMW2 have been shownto mediate adhesion of NTHi to epithelial cells (16). Another family ofhigh molecular weight proteins has been identified in NTHi strains thatlack proteins belonging to HMW1/HMW2 family. The NTHi 115 kDa Hiaprotein (17) is highly similar to the Hsf adhesin expressed by H.influenzae type b strains (18). Another protein, the Hap protein showssimilarity to IgA1 serine proteases and has been shown to be involved inboth adhesion and cell entry (19).

Five major outer membrane proteins (OMP) have been identified andnumerically numbered.

Original studies using H. influenzae type b strains showed thatantibodies specific for P1 and P2 protected infant rats from subsequentchallenge (20-21). P2 was found to be able to induce bactericidal andopsonic antibodies, which are directed against the variable regionspresent within surface exposed loop structures of this integral OMP(22-23). The lipoprotein P4 also could induce bactericidal antibodies(24).

P6 is a conserved peptidoglycan-associated lipoprotein making up 1-5% ofthe outer membrane (25). Later a lipoprotein of about the same mol. wt.was recognized, called PCP (P6 crossreactive protein) (26). A mixture ofthe conserved lipoproteins P4, P6 and PCP did not reveal protection asmeasured in a chinchilla otitis-media model (27). P6 alone appears toinduce protection in the chinchilla model (28).

P5 has sequence homology to the integral Escherichia coli OmpA (29-30).P5 appears to undergo antigenic drift during persistent infections withNTHi (31). However, conserved regions of this protein induced protectionin the chinchilla model of otitis media.

In line with the observations made with gonococci and meningococci, NTHiexpresses a dual human transferrin receptor composed of ThpA and TbpBwhen grown under iron limitation. Anti-TbpB protected infant rats. (32).Hemoglobin/haptoglobin receptors have also been described for NTHi (33).A receptor for Haem: Hemopexin has also been identified (34). Alactoferrin receptor is also present in NTHi, but is not yetcharacterized (35).

A 80 kDa OMP, the D15 surface antigen, provides protection against NTHiin a mouse challenge model. (36). A 42 kDa outer membrane lipoprotein,LPD is conserved amongst Haemophilus influenzae and induces bactericidalantibodies (37). A minor 98 kDa OMP (38), was found to be a protectiveantigen, this OMP may very well be one of the Fe-limitation inducibleOMPs or high molecular weight adhesins that have been characterized. H.influenzae produces IgA1-protease activity (39). IgA1-proteases of NTHireveals a high degree of antigenic variability (40).

Another OMP of NTHi, OMP26, a 26-kDa protein has been shown to enhancepulmonary clearance in a rat model (41). The NTHi HtrA protein has alsobeen shown to be a protective antigen. Indeed, this protein protectedChinchilla against otitis media and protected infant rats against H.influenzae type b bacteremia (42)

BACKGROUND REFERENCES

-   1. Klein, J O (1994) Clin. Inf. Dis 19:823-   2. Murphy, T F (1996) Microbiol. Rev. 60:267-   3. Dickinson, D P et al. (1988) J. Infect. Dis. 158:205-   4. Faden, H L et al. (1991) Ann. Otorhinol. Laryngol. 100:612-   5. Faden, H L et al (1994) J. Infect. Dis. 169:1312-   6. Leach, A J et al. (1994) Pediatr. Infect. Dis. J. 13:983-   7. Prellner, K P et al. (1984) Acta Otolaryngol. 98:343-   8. Stenfors, L-E and Raisanen, S. (1992) J. Infect. Dis. 165:1148-   9. Stenfors, L-E and Raisanen, S. (1994) Acta Otolaryngol. 113:191-   10. Read, R C. et al. (1991) J. Infect. Dis. 163:549-   11. Brinton, C C. et al. (1989) Pediatr. Infect. Dis. J. 8:S54-   12. Kar, S. et al. (1990) Infect. Immun. 58:903-   13. Gildorf, J R. et al. (1992) Infect. Immun. 60:374-   14. St. Geme, J W et al. (1991) Infect. Immun. 59:3366-   15. St. Geme, J W et al. (1993) Infect. Immun. 61: 2233-   16. St. Geme, J W. et al. (1993) Proc. Natl. Acad. Sci. USA 90:2875-   17. Barenkamp, S J. et J W St Geme (1996) Mol. Microbiol. (In press)-   18. St. Geme, J W. et al. (1996) J. Bact. 178:6281-   19. St. Geme, J W. et al. (1994) Mol. Microbiol. 14:217-   20. Loeb, M R. et al. (1987) Infect. Immun. 55:2612-   21. Musson, R S. Jr. et al. (1983) J. Clin. Invest. 72:677-   22. Haase, E M. et al. (1994) Infect. Immun. 62:3712-   23. Troelstra, A. et al. (1994) Infect. Immun. 62:779-   24. Green, B A. et al. (1991) Infect. Immun. 59:3191-   25. Nelson, M B. et al. (1991) Infect. Immun. 59:2658-   26. Deich, R M. et al. (1990) Infect. Immun. 58:3388-   27. Green, B A. et al. (1993) Infect. immun. 61:1950-   28. Demaria, T F. et al. (1996) Infect. Immun. 64:5187-   29. Miyamoto, N., Bakaletz, LO (1996) Microb. Pathog. 21:343.-   30. Munson, R S j.r. et al. (1993) Infect. Immun. 61:1017-   31. Duim, B. et al. (1997) Infect. Immun. 65:1351-   32. Loosmore, S M. et al(1996) Mol. Microbiol. 19:575-   33. Maciver, I. et al. (1996) Infect. Immun. 64:3703-   34. Cope, L D. et al. (1994) Mol. Microbiol. 13:868-   35. Schryvers, A B. et al. (1989) J. Med. Microbiol. 29:121-   36. Flack, F S. et al. (1995) Gene 156:97-   37. Akkoyunlu, M. et al. (1996) Infect. Immun. 64:4586-   38. Kimura, A. et al. (1985) Infect. Immun. 47:253-   39. Mulks, M H. et Shoberg, R J (1994) Meth. Enzymol. 235:543-   40. Lomholt, H. Alphen, Lv, Kilian, M. (1993) Infect. Immun. 61:4575-   41. Kyd, J. M. and Cripps, A. W. (1998) Infect. Immun. 66:2272-   42. Loosmore, S. M. et al. (1998) Infect. Immun. 66:899

The frequency of NTHi infections has risen dramatically in the past fewdecades. This phenomenon has created an unmet medical need for newanti-microbial agents, vaccines, drug screening methods and diagnostictests for this organism. The present invention aims to meet that need.

SUMMARY OF THE INVENTION

The present invention relates to BASB231, in particular BASB231polypeptides and BASB231 polynucleotides, recombinant materials andmethods for their production. In another aspect, the invention relatesto methods for using such polypeptides and polynucleotides, includingprevention and treatment of microbial diseases, amongst others. In afurther aspect, the invention relates to diagnostic assays for detectingdiseases associated with microbial infections and conditions associatedwith such infections, such as assays for detecting expression oractivity of BASB231 polynucleotides or polypeptides.

Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following descriptions and from reading the otherparts of the present disclosure.

DESCRIPTION OF THE INVENTION

The invention relates to BASB231 polypeptides and polynucleotides asdescribed in greater detail below. In particular, the invention relatesto polypeptides and polynucleotides of BASB231 of non typeable H.influenzae.

The invention relates especially to BASB231 polynucleotides and encodedpolypeptides listed in table 1. Those polynucleotides and encodedpolypeptides have the nucleotide and amino acid sequences set out in SEQID NO:1 to SEQ ID NO:74 as described in table 1. TABLE 1 SEQ SEQ LengthLength ID ID Name (nT) (aa) nucl. prot. Description Orf1 453 150 1 2 LOSbiosynthesis enzyme lbga, Haemophilus ducreyi (62%) Orf2 1032 343 3 4Putative d-glycero-d-manno-heptosyl transferase, Actinobacilluspleuropneumoniae (51%) Orf3 813 270 5 6 Formamidopyrimidine-dnaglycosylase, Haemophilus influenzae (74%) Orf4 726 241 7 8 MolybdenumABC transporter, periplasmic molybdate- binding protein, Deinococcusradiodurans (26%) Orf5 741 246 9 10 ABC transporter, Haemophilusinfluenzae (38%) Orf6 1023 340 11 12 ABC transporter, Haemophilusinfluenzae (45%) Orf7 942 313 13 14 ABC transporter, Haemophilusinfluenzae (56%) Orf8 558 185 15 16 Invasin precursor (YadA c-term),Yersinia enterocolitica (27%) Orf9 2373 790 17 18 DNA methylase hsdm,Vibrio cholerae (70%) Orf10 818 272 19 20 Leucyl tRNA synthetase,Borrelia burgdorferi (28%) Orf11 636 211 21 22 ATP dependant DNAhelicase, Deinococcus radiodurans (37%) Orf12 1257 418 23 24 Type Irestriction-modification system (s subunit), Caulobacter crescentus(29%) Orf13 3027 1008 25 26 Type I restriction enzyme hsdr, Vibriocholerae (65%) Orf14 2052 683 27 28 Probable aaa family atpase,Campylobacter jejuni (33%) Orf15 975 324 29 30 No homology with knownprotein Orf16 744 247 31 32 Hypothetical 29.0 kd protein, Aquifexaeolicus (24%) Orf17 846 271 33 34 Hypothetical 27.0 kd protein, Aquifexaeolicus (30%) Orf18 273 90 35 36 Cell division protein ftsk (C-term),Escherichia coli (46%) Orf19 1023 340 37 38 Putative dna-bindingprotein, Neisseria meningitidis (45%) Orf20 711 236 39 40 Hypothetical22.9 kd protein, Actinobacillus actinomycetemcomitans (79%) Orf21 456151 41 42 Yors protein, Bacillus subtilis (26%) Orf22 441 146 43 44Phosphate transport atp-binding protein pstb homolog, Mycoplasmagenitalium (24%) Orf23 642 213 45 46 No homology with known proteinOrf24 1344 447 47 48 Type I restriction protein, Haemophilus influenzae(40%) Orf25 1995 664 49 50 Hypothetical 84.7 kda protein, Thermotogamaritima (25%) Orf26 1155 384 51 52 Anticodon nuclease, Neisseriameningitidis (61%) Orf27 999 332 53 54 wkue. gp8 protein, wolbachia sp.(40%) Orf28 819 272 55 56 Putative transposase protein, Rhizobiummeliloti (40%) Orf29 333 110 57 58 Partial sequence of Bacteriophageif1. orf348 (35%) Orf30 261 86 59 60 Putative cytoplasmic protein,Salmonella typhimurium lt2 (27%) Orf31 927 308 61 62 Tryptophan2-monooxygenase, Agrobacterium tumefaciens (29%) Orf32 315 104 63 64Modification methylase bepi, Brevibacterium epidermidis (51%) Orf33 1464487 65 66 PTS permease for n-acetylglucosamine and Glucose, Vibriofurnissii (71%) Orf34 888 295 67 68 Putative lysr-family transcriptionalregulator, Neisseria meningitidis (91%) Orf35 843 280 69 70 Hypothetical118.9 kda protein, Plasmodium falciparum (19%) Orf36 393 130 71 72tiorf34 protein, Agrobacterium tumefaciens (ti plasmid ptit37) (25%)Orf37 675 224 73 74 Modification methylase bepi, Brevibacteriumepidermidis (55%)

BASB231 polypeptides and polynucleotides are specific to non typeable H.influenzae (they are not present in H. influenzae Rd strain), and arethus particularly useful in the ntHi diagnostic field, as a whole hostof ntHi-specific DNA probes and ntHi-specific enzyme functionalities maybe used to detect the presence of ntHi in a sample as distinct fromencapsuated Hi strains.

In addition, the availability of the above sequences allows: a) theupregulation or downregulation (i.e. knock-out of functional expression)of any of the above genes to create an ntHi strain with novelcharacteristics; b) the insertion and expression of any of the abovegenes in a non-ntHi host to introduce a ntHi-specific functionality intosaid host; and c) the purification of an ntHi-specific enzyme from theabove list for performing in vitro reactions. To knock-out a gene, thegene (or a portion thereof) may be deleted, or may have an insertion orother mutation, or may have its promoter removed or replaced, such thatexpression of a gene product with the wild-type functionality issubstantially (preferably completely) switched off. For instance Orf1encodes a Lipo-oligosaccharide (LOS) biosynthesis enzyme (responsiblefor adding sugar groups to the antigenic ntHi-specific LOS molecule).With the Orf1 gene and protein sequences a skilled person will readilybe able to ensure the above enzyme is either constitutively expressed orpermanently switched off in a mutant ntHi strain in order to obtain amore consistent or a different LOS structure (respectively) which may beadvantageously used for vaccine puroposes (either as LOS complexed withntHi outer membrane, or as purified LOS). In addition the enzyme may beisolated or recombinantly produced for its specific function to be usedin vitro to produce novel synthetic oligosaccharide structures.

It is understood that sequences recited in the Sequence Listing below as“DNA” represent an exemplification of one embodiment of the invention,since those of ordinary skill will recognize that such sequences can beusefully employed in polynucleotides in general, includingribopolynucleotides.

The sequences of the BASB231 polynucleotides are set out in SEQ ID NO:1,3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73. SEQGroup 1 refers herein to any one of the polynucleotides set out in SEQID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71or 73. The sequences of the BASB231 encoded polypeptides are set out inSEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68,70, 72. SEQ Group 2 refers herein to any one of the encoded polypeptidesset out in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64,66, 68, 70 or 72.

Polypeptides

In one aspect of the invention there are provided polypeptides of nontypeable H. influenzae referred to herein as “BASB231” and “BASB231polypeptides” as well as biologically, diagnostically, prophylactically,clinically or therapeutically useful variants thereof, and compositionscomprising the same.

The present invention further provides for:

-   (a) an isolated polypeptide which comprises an amino acid sequence    which has at least 85% identity, preferably at least 90% identity,    more preferably at least 95% identity, most preferably at least    97-99% or exact identity, to that of any sequence of SEQ Group 2;-   (b) a polypeptide encoded by an isolated polynucleotide comprising a    polynucleotide sequence which has at least 85% identity, preferably    at least 90% identity, more preferably at least 95% identity, even    more preferably at least 97-99% or exact identity to any sequence of    SEQ Group 1 over the entire length of the selected sequence of SEQ    Group 1; or-   (c) a polypeptide encoded by an isolated polynucleotide comprising a    polynucleotide sequence encoding a polypeptide which has at least    85% identity, preferably at least 90% identity, more preferably at    least 95% identity, even more preferably at least 97-99% or exact    identity, to the amino acid sequence of any sequence of SEQ Group 2.

The BASB231 polypeptides provided in SEQ Group 2 are the BASB231polypeptides from non typeable H. influenzae strain ATCC PTA-1816.

The invention also provides an immunogenic (or enzymatically functional)fragment of a BASB231 polypeptide, that is, a contiguous portion of theBASB231 polypeptide which has the same or substantially the sameimmunogenic activity (or enzymatic activity) as the polypeptidecomprising the corresponding amino acid sequence selected from SEQ Group2; That is to say, the fragment (if necessary when coupled to a carrier)is capable of raising an immune response which recognises the BASB231polypeptide (or can perform the same enzymatic function as the BASB231polypeptide). Such an immunogenic (or enzymatically functional) fragmentmay include, for example, the BASB231 polypeptide lacking an N-terminalleader sequence, and/or a transmembrane domain and/or a C-terminalanchor domain. In a preferred aspect the immunogenic (or enzymaticallyfunctional) fragment of BASB231 according to the invention comprisessubstantially all of the extracellular domain of a polypeptide which hasat least 85% identity, preferably at least 90% identity, more preferablyat least 95% identity, most preferably at least 97-99% identity, to thata sequence selected from SEQ Group 2 over the entire length of saidsequence.

A fragment is a polypeptide having an amino acid sequence that isentirely the same as part but not all of any amino acid sequence of anypolypeptide of the invention. As with BASB231 polypeptides, fragmentsmay be “free-standing,” or comprised within a larger polypeptide ofwhich they form a part or region, most preferably as a single continuousregion in a single larger polypeptide.

Preferred fragments include, for example, truncation polypeptides havinga portion of an amino acid sequence selected from SEQ Group 2 or ofvariants thereof, such as a continuous series of residues that includesan amino- and/or carboxyl-terminal amino acid sequence. Degradationforms of the polypeptides of the invention produced by or in a hostcell, are also preferred. Further preferred are fragments characterizedby structural or functional attributes such as fragments that comprisealpha-helix and alpha-helix forming regions, beta-sheet andbeta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions.

Further preferred fragments include an isolated polypeptide comprisingan amino acid sequence having at least 15, 20, 30, 40, 50 or 100contiguous amino acids from an amino acid sequence selected from SEQGroup 2 or an isolated polypeptide comprising an amino acid sequencehaving at least 15, 20, 30, 40, 50 or 100 contiguous amino acidstruncated or deleted from an amino acid sequence selected from SEQ Group2.

Still further preferred fragments are those which comprise a B-cell orT-helper epitope, for example those fragments/peptides readilydetermined from the SEQ Group 2 sequences by well known predictionalgorithms. The B-cell epitopes of a protein are mainly localized at itssurface. To predict B-cell epitopes of BASB231 polypeptides two methodscan be combined: 2D-structure prediction and antigenic index prediction.The 2D-structure prediction can be made using the Chou Fasman method(from Chou P Y and Fasman G D, Biochemistry, vol 13(2), pp 222-245,1974) and the Gor method (from Gamier J, Osguthorpe D J and Robson B, JMol biol vol 120(1), pp97-120, 1978). The antigenic index can becalculated on the basis of the method described by Jameson and Wolf(CABIOS 4:181-186 [1988]). The parameters used in this program are theantigenic index and the minimal length for an antigenic peptide. Anantigenic index of 0.9 for a minimum of 5 consecutive amino acids ispreferably used as threshold in the program. Peptides comprisingpotential B-cell epitopes can be useful (preferably conjugated orrecombinantly joined to a larger protein) in a vaccine composition forthe prevention of ntHi infections, and typically comprise 5 or more(e.g. 6, 7, 8, 9, 10, 11, 12, 15 or 20) contiguous amino acids from theBASB231 polypeptide sequence which can elicit an immune response in ahost against the BASB231 polypeptide.

T-helper cell epitopes are peptides bound to HLA class II molecules andrecognized by T-helper cells. The prediction of useful T-helper cellepitopes of BASB231 polypeptide is preferably based on the TEPITOPEmethod described by Sturniolo at al. (Nature Biotech. 17: 555-561[1999]). Peptides comprising potential T-cell epitopes can be useful(preferably conjugated to peptides, polypeptides or polysaccharides) forvaccine purposes, and typically comprise 5 or more (e.g. 6, 7, 8, 9, 10,11, 12, 14, 16, 18, 20, 23, 26 or 30) contiguous amino acids from theBASB231 polypeptide sequence which preserve an effective T-helperepitope from BASB231 polypeptides.

Fragments of the polypeptides of the invention may be employed forproducing the corresponding full-length polypeptide by peptidesynthesis; therefore, these fragments may be employed as intermediatesfor producing the full-length polypeptides of the invention.

Particularly preferred are variants in which several, 5-10, 1-5,1-3, 1-2or 1 amino acids are substituted, deleted, or added in any combination.

The polypeptides, or immunogenic (or enzymatically functional)fragments, of the invention may be in the form of the “mature” proteinor may be a part of a larger protein such as a precursor or a fusionprotein. It is often advantageous to include an additional amino acidsequence which contains secretory or leader sequences, pro-sequences,sequences which aid in purification such as multiple histidine residues,or an additional sequence for stability during recombinant production.Furthermore, addition of exogenous polypeptide or lipid tail orpolynucleotide sequences to increase the immunogenic potential of thefinal molecule is also considered.

In one aspect, the invention relates to genetically engineered solublefusion proteins comprising a polypeptide of the present invention, or afragment thereof, and various portions of the constant regions of heavyor light chains of immunoglobulins of various subclasses (IgG, IgM, IgA,IgE). Preferred as an immunoglobulin is the constant part of the heavychain of human IgG, particularly IgG1, where fusion takes place at thehinge region. In a particular embodiment, the Fc part can be removedsimply by incorporation of a cleavage sequence which can be cleaved withblood clotting factor Xa.

Furthermore, this invention relates to processes for the preparation ofthese fusion proteins by genetic engineering, and to the use thereof fordrug screening, diagnosis and therapy. A further aspect of the inventionalso relates to polynucleotides encoding such fusion proteins. Examplesof fusion protein technology can be found in International PatentApplication Nos. WO94/29458 and WO94/22914.

The proteins may be chemically conjugated, or expressed as recombinantfusion proteins allowing increased levels to be produced in anexpression system as compared to non-fused protein. The fusion partnermay assist in providing T helper epitopes (immunological fusionpartner), preferably T helper epitopes recognised by humans, or assistin expressing the protein (expression enhancer) at higher yields thanthe native recombinant protein. Preferably the fusion partner will beboth an immunological fusion partner and expression enhancing partner.

Fusion partners include protein D from Haemophilus influenzae and thenon-structural protein from influenza virus, NS1 (hemagglutinin).Another fusion partner is the protein known as Omp26 (WO 97/01638).Another fusion partner is the protein known as LytA. Preferably the Cterminal portion of the molecule is used. LytA is derived fromStreptococcus pneumoniae which synthesize an N-acetyl-L-alanine amidase,amidase LytA, (coded by the lytA gene {Gene, 43 (1986) page 265-272}) anautolysin that specifically degrades certain bonds in the peptidoglycanbackbone. The C-terminal domain of the LytA protein is responsible forthe affinity to the choline or to some choline analogues such as DEAE.This property has been exploited for the development of E. coli C-LytAexpressing plasmids useful for expression of fusion proteins.Purification of hybrid proteins containing the C-LytA fragment at itsamino terminus has been described {Biotechnology: 10, (1992) page795-798}. It is possible to use the repeat portion of the LytA moleculefound in the C terminal end starting at residue 178, for exampleresidues 188-305.

The present invention also includes variants of the aforementionedpolypeptides, that is polypeptides that vary from the referents byconservative amino acid substitutions, whereby a residue is substitutedby another with like characteristics. Typical such substitutions areamong Ala, Val, Leu and Ile; among Ser and Thr; among the acidicresidues Asp and Glu; among Asn and Gln; and among the basic residuesLys and Arg; or aromatic residues Phe and Tyr.

Polypeptides of the present invention can be prepared in any suitablemanner. Such polypeptides include isolated naturally occurringpolypeptides, recombinantly produced polypeptides, syntheticallyproduced polypeptides, or polypeptides produced by a combination ofthese methods. Means for preparing such polypeptides are well understoodin the art.

It is most preferred that a polypeptide of the invention is derived fromnon typeable H. influenzae, however, it may preferably be obtained fromother organisms of the same taxonomic genus. A polypeptide of theinvention may also be obtained, for example, from organisms of the sametaxonomic family or order.

Polynucleotides

It is an object of the invention to provide polynucleotides that encodeBASB231 polypeptides, particularly polynucleotides that encode thepolypeptides herein designated BASB231.

In a particularly preferred embodiment of the invention thepolynucleotides comprise a region encoding BASB231 polypeptidescomprising sequences set out in SEQ Group 1 which include full lengthgene, or a variant thereof.

The BASB231 polynucleotides provided in SEQ Group I are the BASB231polynucleotides from non typeable H. influenzae strain ATCC PTA-1816.

As a further aspect of the invention there are provided isolated nucleicacid molecules encoding and/or expressing BASB231 polypeptides andpolynucleotides, particularly non typeable H. influenzae BASB231polypeptides and polynucleotides, including, for example, unprocessedRNAs, ribozyme RNAs, mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs. Furtherembodiments of the invention include biologically, diagnostically,prophylactically, clinically or therapeutically useful polynucleotidesand polypeptides, and variants thereof, and compositions comprising thesame.

Another aspect of the invention relates to isolated polynucleotides,including at least one full length gene, that encodes a BASB231polypeptide having a deduced amino acid sequence of SEQ Group 2 andpolynucleotides closely related thereto and variants thereof.

In another particularly preferred embodiment of the invention relates toBASB231 polypeptide from non typeable H. influenzae comprising orconsisting of an amino acid sequence selected from SEQ Group 2 or avariant thereof.

Using the information provided herein, such as a polynucleotidesequences set out in SEQ Group 1, a polynucleotide of the inventionencoding BASB231 polypeptides may be obtained using standard cloning andscreening methods, such as those for cloning and sequencing chromosomalDNA fragments from bacteria using non typeable H. influenzae strain3224Acells as starting material, followed by obtaining a full length clone.For example, to obtain a polynucleotide sequence of the invention, suchas a polynucleotide sequence given in SEQ Group 1, typically a libraryof clones of chromosomal DNA of non typeable H. influenzae strain 3224Ain E. coli or some other suitable host is probed with a radiolabeledoligonucleotide, preferably a 17-mer or longer, derived from a partialsequence. Clones carrying DNA identical to that of the probe can then bedistinguished using stringent hybridization conditions. By sequencingthe individual clones thus identified by hybridization with sequencingprimers designed from the original polypeptide or polynucleotidesequence it is then possible to extend the polynucleotide sequence inboth directions to determine a full length gene sequence. Conveniently,such sequencing is performed, for example, using denatured doublestranded DNA prepared from a plasmid clone. Suitable techniques aredescribed by Maniatis, T., Fritsch, E. F. and Sambrook et al., MOLECULARCLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989). (see in particular Screening ByHybridization 1.90 and Sequencing Denatured Double-Stranded DNATemplates 13.70). Direct genomic DNA sequencing may also be performed toobtain a full length gene sequence. Illustrative of the invention, eachpolynucleotide set out in SEQ Group 1 was discovered in a DNA libraryderived from non typeable H. influenzae.

Moreover, each DNA sequence set out in SEQ Group 1 contains an openreading frame encoding a protein having about the number of amino acidresidues set forth in SEQ Group 2 with a deduced molecular weight thatcan be calculated using amino acid residue molecular weight values wellknown to those skilled in the art.

The polynucleotides of SEQ Group 1, between the start codon and the stopcodon, encode respectively the polypeptides of SEQ Group 2. Thenucleotide number of start codon and first nucleotide of stop codon arelisted in table 2 for each polynucleotide of SEQ Group 1. TABLE 2 1^(st)nucleotide of Name Start codon Stop codon Orf1 1 453 Orf2 1 1030 Orf3 1811 Orf4 1 724 Orf5 1 739 Orf6 1 1021 Orf7 1 940 Orf8  1* 556 Orf9 12371 Orf10 1 816 Orf11 1 634 Orf12 1 1255 Orf13 1 3025 Orf14 1 2050Orf15 1 973 Orf16  1* 742 Orf17 1 814 Orf18  1* 271 Orf19 1 1021 Orf20 1709 Orf21 1 454 Orf22  1* 439 Orf23 1 642 Orf24 1 1342 Orf25 1 1993Orf26  1* 1153 Orf27 1 997 Orf28 1 817 Orf29  1* 331 Orf30 1 259 Orf31 1916 Orf32  1* 310 Orf33 1 1462 Orf34 1 886 Orf35  1* 841 Orf36  1* 391Orf37 1 673*It is not the start codon but it is the first nucleotide of the codingsequence

In a further aspect, the present invention provides for an isolatedpolynucleotide comprising or consisting of:

-   (a) a polynucleotide sequence which has at least 85% identity,    preferably at least 90% identity, more preferably at least 95%    identity, even more preferably at least 97-99% or exact identity, to    any polynucleotide sequence from SEQ Group 1 over the entire length    of the polynucleotide sequence from SEQ Group 1; or    -   (b) a polynucleotide sequence encoding a polypeptide which has        at least 85% identity, preferably at least 90% identity, more        preferably at least 95% identity, even more preferably at least        97-99% or 100% exact identity, to any amino acid sequence        selected from SEQ Group 2, over the entire length of the amino        acid sequence from SEQ Group 2.

A polynucleotide encoding a polypeptide of the present invention,including homologs and orthologs from species other than non typeable H.influenzae, may be obtained by a process which comprises the steps ofscreening an appropriate library under stringent hybridizationconditions (for example, using a temperature in the range of 45-65° C.and an SDS concentration from 0.1-1%) with a labeled or detectable probeconsisting of or comprising any sequence selected from SEQ Group 1 or afragment thereof; and isolating a full-length gene and/or genomic clonescontaining said polynucleotide sequence.

The invention provides a polynucleotide sequence identical over itsentire length to a coding sequence (open reading frame) set out in SEQGroup 1. Also provided by the invention is a coding sequence for amature polypeptide or a fragment thereof, by itself as well as a codingsequence for a mature polypeptide or a fragment in reading frame withanother coding sequence, such as a sequence encoding a leader orsecretory sequence, a pre-, or pro- or prepro-protein sequence. Thepolynucleotide of the invention may also contain at least one non-codingsequence, including for example, but not limited to at least onenon-coding 5′ and 3′ sequence, such as the transcribed butnon-translated sequences, termination signals (such as rho-dependent andrho-independent termination signals), ribosome binding sites, Kozaksequences, sequences that stabilize mRNA, introns, and polyadenylationsignals. The polynucleotide sequence may also comprise additional codingsequence encoding additional amino acids. For example, a marker sequencethat facilitates purification of the fused polypeptide can be encoded.In certain embodiments of the invention, the marker sequence is ahexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) anddescribed in Gentz et al., Proc. Natl. Acad. Sci., USA 86: 821-824(1989), or an HA peptide tag (Wilson et al, Cell 37: 767 (1984), both ofwhich may be useful in purifying polypeptide sequence fused to them.Polynucleotides of the invention also include, but are not limited to,polynucleotides comprising a structural gene and its naturallyassociated sequences that control gene expression.

The nucleotide sequence encoding the BASB231 polypeptide of SEQ Group 2may be identical to the corresponding polynucleotide encoding sequenceof SEQ Group 1. The position of the first and last nucleotides of theencoding sequences of SEQ Goup 1 are listed in table 3. Alternatively itmay be any sequence, which as a result of the redundancy (degeneracy) ofthe genetic code, also encodes a polypeptide of SEQ Group 2. TABLE 3Name Start codon Last nucleotide encoding polypeptide Orf1 1 452 Orf2 11029 Orf3 1 810 Orf4 1 723 Orf5 1 738 Orf6 1 1020 Orf7 1 939 Orf8  1*555 Orf9 1 2370 Orf10 1 815 Orf11 1 633 Orf12 1 1254 Orf13 1 3024 Orf141 2049 Orf15 1 972 Orf16  1* 741 Orf17 1 813 Orf18  1* 270 Orf19 1 1020Orf20 1 708 Orf21 1 453 Orf22  1* 438 Orf23 1 641 Orf24 1 1341 Orf25 11992 Orf26  1* 1152 Orf27 1 996 Orf28 1 816 Orf29  1* 330 Orf30 1 258Orf31 1 915 Orf32  1* 309 Orf33 1 1461 Orf34 1 885 Orf35  1* 840 Orf36 1* 390 Orf37 1 672*It is not the start codon but it is the first nucleotide of the codingsequence

The term “polynucleotide encoding a polypeptide” as used hereinencompasses polynucleotides that include a sequence encoding apolypeptide of the invention, particularly a bacterial polypeptide andmore particularly a polypeptide of the non typeable H. influenzaeBASB231 having an amino acid sequence set out in any of the sequences ofSEQ Group 2. The term also encompasses polynucleotides that include asingle continuous region or discontinuous regions encoding thepolypeptide (for example, polynucleotides interrupted by integratedphage, an integrated insertion sequence, an integrated vector sequence,an integrated transposon sequence, or due to RNA editing or genomic DNAreorganization) together with additional regions, that also may containcoding and/or non-coding sequences.

The invention further relates to variants of the polynucleotidesdescribed herein that encode variants of a polypeptide having a deducedamino acid sequence of any of the sequences of SEQ Group 2. Fragments ofpolynucleotides of the invention may be used, for example, to synthesizefull-length polynucleotides of the invention.

Further particularly preferred embodiments are polynucleotides encodingBASB231 variants, that have the amino acid sequence of BASB231polypeptide of any sequence from SEQ Group 2 in which several, a few, 5to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted,modified, deleted and/or added, in any combination. Especially preferredamong these are silent substitutions, additions and deletions, that donot alter the properties and activities of BASB231 polypeptide.

Further preferred embodiments of the invention are polynucleotides thatare at least 85% identical over their entire length to a polynucleotideencoding BASB231 polypeptide having an amino acid sequence set out inany of the sequences of SEQ Group 2, and polynucleotides that arecomplementary to such polynucleotides. Alternatively, most highlypreferred are polynucleotides that comprise a region that is at least90% identical over its entire length to a polynucleotide encodingBASB231 polypeptide and polynucleotides complementary thereto. In thisregard, polynucleotides at least 95% identical over their entire lengthto the same are particularly preferred. Furthermore, those with at least97% are highly preferred among those with at least 95%, and among thesethose with at least 98% and at least 99% are particularly highlypreferred, with at least 99% being the more preferred.

Preferred embodiments are polynucleotides encoding polypeptides thatretain substantially the same biological function or activity as themature polypeptide encoded by a DNA sequence selected from SEQ Group 1.

In accordance with certain preferred embodiments of this invention thereare provided polynucleotides that hybridize, particularly understringent conditions, to BASB231 polynucleotide sequences, such as thosepolynucleotides of SEQ Group 1.

The invention further relates to polynucleotides that hybridize to thepolynucleotide sequences provided herein. In this regard, the inventionespecially relates to polynucleotides that hybridize under stringentconditions to the polynucleotides described herein. As herein used, theterms “stringent conditions” and “stringent hybridization conditions”mean hybridization occurring only if there is at least 95% andpreferably at least 97% identity between the sequences. A specificexample of stringent hybridization conditions is overnight incubation at42° C. in a solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml of denatured,sheared salmon sperm DNA, followed by washing the hybridization supportin 0.1×SSC at about 65° C. Hybridization and wash conditions are wellknown and exemplified in Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989),particularly Chapter 11 therein. Solution hybridization may also be usedwith the polynucleotide sequences provided by the invention.

Such polynucleotides preferably have at least 15 or 30 nucleotideresidues or base pairs and may have at least 50 nucleotide residues orbase pairs. Particularly preferred polynucleotides will have at least 20nucleotide residues or base pairs and will have less than 30 nucleotideresidues or base pairs. Most preferably these polynucleotides arecontiguous polynucleotides from a BASB231 polynucleotide sequence. Suchpolynucleotides are particularly useful in diagnostic methods where thespecific hybridisation of these polynucleotides to the ntHi genome candifferentiate the presence of ntHi in a sample rather than that ofencapsulated Hi strains.

The invention also provides a polynucleotide consisting of or comprisinga polynucleotide sequence obtained by screening an appropriate librarycontaining the complete gene for a polynucleotide sequence set forth inany of the sequences of SEQ Group 1 under stringent hybridizationconditions with a probe having the sequence of said polynucleotidesequence set forth in the corresponding sequence of SEQ Group 1 or afragment thereof; and isolating said polynucleotide sequence. Fragmentsuseful for obtaining such a polynucleotide include, for example, probesand primers fully described elsewhere herein.

As discussed elsewhere herein regarding polynucleotide assays of theinvention, for instance, the polynucleotides of the invention, may beused as a hybridization probe for RNA, cDNA and genomic DNA to isolatefull-length cDNAs and genomic clones encoding BASB231 and to isolatecDNA and genomic clones of other genes that have a high identity,particularly high sequence identity, to the BASB231 gene. Such probesgenerally will comprise at least 15 nucleotide residues or base pairs.Preferably, such probes will have at least 30 nucleotide residues orbase pairs and may have at least 50 nucleotide residues or base pairs.Particularly preferred probes will have at least 20 nucleotide residuesor base pairs and will have less than 30 nucleotide residues or basepairs.

A coding region of a BASB231 gene may be isolated by screening using aDNA sequence provided in SEQ Group 1 to synthesize an oligonucleotideprobe. A labeled oligonucleotide having a sequence complementary to thatof a gene of the invention is then used to screen a library of cDNA,genomic DNA or mRNA to determine which members of the library the probehybridizes to.

There are several methods available and well known to those skilled inthe art to obtain full-length DNAs, or extend short DNAs, for examplethose based on the method of Rapid Amplification of cDNA ends (RACE)(see, for example, Frohman, et al., PNAS USA 85: 8998-9002, 1988).Recent modifications of the technique, exemplified by the Marathon™technology (Clontech Laboratories Inc.) for example, have significantlysimplified the search for longer cDNAs. In the Marathon™ technology,cDNAs have been prepared from mRNA extracted from a chosen tissue and an‘adaptor’ sequence ligated onto each end. Nucleic acid amplification(PCR) is then carried out to amplify the “missing” 5′ end of the DNAusing a combination of gene specific and adaptor specificoligonucleotide primers. The PCR reaction is then repeated using“nested” primers, that is, primers designed to anneal within theamplified product (typically an adaptor specific primer that annealsfurther 3′ in the adaptor sequence and a gene specific primer thatanneals further 5′ in the selected gene sequence). The products of thisreaction can then be analyzed by DNA sequencing and a full-length DNAconstructed either by joining the product directly to the existing DNAto give a complete sequence, or carrying out a separate full-length PCRusing the new sequence information for the design of the 5′ primer.

The polynucleotides and polypeptides of the invention may be employed,for example, as research reagents and materials for discovery oftreatments of and diagnostics for diseases, particularly human diseases,as further discussed herein relating to polynucleotide assays. Thepolynucleotides of the invention that are oligonucleotides derived froma sequence of SEQ Group 1 may be used in the processes herein asdescribed, but preferably for PCR, to determine whether or not thepolynucleotides identified herein in whole or in part are transcribed inbacteria in infected tissue. It is recognized that such sequences willalso have utility in diagnosis of the stage of infection and type ofinfection the pathogen has attained.

The invention also provides polynucleotides that encode a polypeptidethat is the mature protein plus additional amino or carboxyl-terminalamino acids, or amino acids interior to the mature polypeptide (when themature form has more than one polypeptide chain, for instance). Suchsequences may play a role in processing of a protein from precursor to amature form, may allow protein transport, may lengthen or shortenprotein half-life or may facilitate manipulation of a protein for assayor production, among other things. As generally is the case in vivo, theadditional amino acids may be processed away from the mature protein bycellular enzymes.

For each and every polynucleotide of the invention there is provided apolynucleotide complementary to it. It is preferred that thesecomplementary polynucleotides are fully complementary to eachpolynucleotide with which they are complementary.

A precursor protein, having a mature form of the polypeptide fused toone or more prosequences may be an inactive form of the polypeptide.When prosequences are removed such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

In addition to the standard A, G, C, T/U representations fornucleotides, the term “N” may also be used in describing certainpolynucleotides of the invention. “N” means that any of the four DNA orRNA nucleotides may appear at such a designated position in the DNA orRNA sequence, except it is preferred that N is not a nucleic acid thatwhen taken in combination with adjacent nucleotide positions, when readin the correct reading frame, would have the effect of generating apremature termination codon in such reading frame.

In sum, a polynucleotide of the invention may encode a mature protein, amature protein plus a leader sequence (which may be referred to as apreprotein), a precursor of a mature protein having one or moreprosequences that are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

In accordance with an aspect of the invention, there is provided the useof a polynucleotide of the invention for therapeutic or prophylacticpurposes, in particular genetic immunization.

The use of a polynucleotide of the invention in genetic immunizationwill preferably employ a suitable delivery method such as directinjection of plasmid DNA into muscles (Wolff et al., Hum Mol Genet(1992) 1: 363, Manthorpe et al., Hum. Gene Ther. (1983) 4: 419),delivery of DNA complexed with specific protein carriers (Wu et al., J.Biol. Chem. (1989) 264: 16985), coprecipitation of DNA with calciumphosphate (Benvenisty & Reshef, PNAS USA, (1986) 83: 9551),encapsulation of DNA in various forms of liposomes (Kaneda et al.,Science (1989) 243: 375), particle bombardment (Tang et al., Nature(1992) 356:152, Eisenbraun et al., DNA Cell Biol (1993) 12: 791) and invivo infection using cloned retroviral vectors (Seeger et al., PNAS USA(1984) 81: 5849).

Vectors, Host Cells, Expression Systems

The invention also relates to vectors that comprise a polynucleotide orpolynucleotides of the invention, host cells that are geneticallyengineered with vectors of the invention and the production ofpolypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the invention.

Recombinant polypeptides of the present invention may be prepared byprocesses well known in those skilled in the art from geneticallyengineered host cells comprising expression systems. Accordingly, in afurther aspect, the present invention relates to expression systems thatcomprise a polynucleotide or polynucleotides of the present invention,to host cells which are genetically engineered with such expressionsystems, and to the production of polypeptides of the invention byrecombinant techniques.

For recombinant production of the polypeptides of the invention, hostcells can be genetically engineered to incorporate expression systems orportions thereof or polynucleotides of the invention. Introduction of apolynucleotide into the host cell can be effected by methods describedin many standard laboratory manuals, such as Davis, et al., BASICMETHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook, et al., MOLECULARCLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989), such as, calcium phosphatetransfection, DEAE-dextran mediated transfection, transvection,microinjection, cationic lipid-mediated transfection, electroporation,conjugation, transduction, scrape loading, ballistic introduction andinfection.

Representative examples of appropriate hosts include bacterial cells,such as cells of streptococci, staphylococci, enterococci, E. coli,streptomyces, cyanobacteria, Bacillus subtilis, Neisseria meningitidis,Haemophilus influenzae and Moraxella catarrhalis; fungal cells, such ascells of a yeast, Kluveromyces, Saccharomyces, Pichia, a basidiomycete,Candida albicans and Aspergillus; insect cells such as cells ofDrosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS, HeLa,C127, 3T3, BHK, 293, CV-1 and Bowes melanoma cells; and plant cells,such as cells of a gymnosperm or angiosperm.

A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among others,chromosomal-, episomal- and virus-derived vectors, for example, vectorsderived from bacterial plasmids, from bacteriophage, from transposons,from yeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses, picomaviruses, retroviruses, and alphaviruses and vectorsderived from combinations thereof, such as those derived from plasmidand bacteriophage genetic elements, such as cosmids and phagemids. Theexpression system constructs may contain control regions that regulateas well as engender expression. Generally, any system or vector suitableto maintain, propagate or express polynucleotides and/or to express apolypeptide in a host may be used for expression in this regard. Theappropriate DNA sequence may be inserted into the expression system byany of a variety of well-known and routine techniques, such as, forexample, those set forth in Sambrook et al., MOLECULAR CLONING, ALABORATORY MANUAL, (supra).

In recombinant expression systems in eukaryotes, for secretion of atranslated protein into the lumen of the endoplasmic reticulum, into theperiplasmic space or into the extracellular environment, appropriatesecretion signals may be incorporated into the expressed polypeptide.These signals may be endogenous to the polypeptide or they may beheterologous signals.

Polypeptides of the present invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, ion metalaffinity chromatography (IMAC) is employed for purification. Well knowntechniques for refolding proteins may be employed to regenerate activeconformation when the polypeptide is denatured during intracellularsynthesis, isolation and or purification.

The expression system may also be a recombinant live microorganism, suchas a virus or bacterium. The gene of interest can be inserted into thegenome of a live recombinant virus or bacterium. Inoculation and in vivoinfection with this live vector will lead to in vivo expression of theantigen and induction of immune responses. Viruses and bacteria used forthis purpose are for instance: poxviruses (e.g; vaccinia, fowipox,canarypox), alphaviruses (Sindbis virus, Semliki Forest Virus,Venezuelian Equine Encephalitis Virus), adenoviruses, adeno-associatedvirus, picomaviruses (poliovirus, rhinovirus), herpesviruses (varicellazoster virus, etc), Listeria, Salmonella, Shigella, BCG, streptococci.These viruses and bacteria can be virulent, or attenuated in variousways in order to obtain live vaccines. Such live vaccines also form partof the invention.

Diagnostic, Prognostic, Serotyping and Mutation Assays

This invention is also related to the use of BASB231 polynucleotides andpolypeptides of the invention for use as diagnostic reagents. Detectionof BASB231 polynucleotides and/or polypeptides in a eukaryote,particularly a mammal, and especially a human, will provide a diagnosticmethod for diagnosis of disease, staging of disease or response of aninfectious organism to drugs. Eukaryotes, particularly mammals, andespecially humans, particularly those infected or suspected to beinfected with an organism comprising the BASB231 gene or protein, may bedetected at the nucleic acid or amino acid level by a variety of wellknown techniques as well as by methods provided herein.

Polypeptides and polynucleotides for prognosis, diagnosis or otheranalysis may be obtained from a putatively infected and/or infectedindividual's bodily materials. Polynucleotides from any of thesesources, particularly DNA or RNA, may be used directly for detection ormay be amplified enzymatically by using PCR or any other amplificationtechnique prior to analysis. RNA, particularly mRNA, cDNA and genomicDNA may also be used in the same ways. Using amplification,characterization of the species and strain of infectious or residentorganism present in an individual, may be made by an analysis of thegenotype of a selected polynucleotide of the organism. Deletions andinsertions can be detected by a change in size of the amplified productin comparison to a genotype of a reference sequence selected from arelated organism, preferably a different species of the same genus or adifferent strain of the same species. Point mutations can be identifiedby hybridizing amplified DNA to labeled BASB231 polynucleotidesequences. Perfectly or significantly matched sequences can bedistinguished from imperfectly or more significantly mismatched duplexesby DNase or RNase digestion, for DNA or RNA respectively, or bydetecting differences in melting temperatures or renaturation kinetics.Polynucleotide sequence differences may also be detected by alterationsin the electrophoretic mobility of polynucleotide fragments in gels ascompared to a reference sequence. This may be carried out with orwithout denaturing agents. Polynucleotide differences may also bedetected by direct DNA or RNA sequencing. See, for example, Myers etal., Science, 230: 1242 (1985). Sequence changes at specific locationsalso may be revealed by nuclease protection assays, such as RNase, V1and S1 protection assay or a chemical cleavage method. See, for example,Cotton et al., Proc. Natl. Acad. Sci., USA, 85: 4397-4401 (1985).

In another embodiment, an array of oligonucleotides probes comprisingBASB231 nucleotide sequence or fragments thereof can be constructed toconduct efficient screening of, for example, genetic mutations,serotype, taxonomic classification or identification. Array technologymethods are well known and have general applicability and can be used toaddress a variety of questions in molecular genetics including geneexpression, genetic linkage, and genetic variability (see, for example,Chee et al., Science, 274: 610 (1996)).

Thus in another aspect, the present invention relates to a diagnostickit which comprises:

-   (a) a polynucleotide of the present invention, preferably any of the    nucleotide sequences of SEQ Group 1, or a fragment thereof;-   (b) a nucleotide sequence complementary to that of (a);-   (c) a polypeptide of the present invention, preferably any of the    polypeptides of SEQ Group 2 or a fragment thereof; or-   (d) an antibody to a polypeptide of the present invention,    preferably to any of the polypeptides of SEQ Group 2.

It will be appreciated that in any such kit, (a), (b), (c) or (d) maycomprise a substantial component. Such a kit will be of use indiagnosing a disease or susceptibility to a Disease, among others.

This invention also relates to the use of polynucleotides of the presentinvention as diagnostic reagents. Detection of a mutated form of apolynucleotide of the invention, preferably any sequence of SEQ Group 1,which is associated with a disease or pathogenicity will provide adiagnostic tool that can add to, or define, a diagnosis of a disease, aprognosis of a course of disease, a determination of a stage of disease,or a susceptibility to a disease, which results from under-expression,over-expression or altered expression of the polynucleotide. Organisms,particularly infectious organisms, carrying mutations in suchpolynucleotide may be detected at the polynucleotide level by a varietyof techniques, such as those described elsewhere herein.

Cells from an organism carrying mutations or polymorphisms (allelicvariations) in a polynucleotide and/or polypeptide of the invention mayalso be detected at the polynucleotide or polypeptide level by a varietyof techniques, to allow for serotyping, for example. For example, RT-PCRcan be used to detect mutations in the RNA. It is particularly preferredto use RT-PCR in conjunction with automated detection systems, such as,for example, GeneScan. RNA, cDNA or genomic DNA may also be used for thesame purpose, PCR. As an example, PCR primers complementary to apolynucleotide encoding BASB231 polypeptide can be used to identify andanalyze mutations.

The invention further provides primers with 1, 2, 3 or 4 nucleotidesremoved from the 5′ and/or the 3′ end. These primers may be used for,among other things, amplifying BASB231 DNA and/or RNA isolated from asample derived from an individual, such as a bodily material. Theprimers may be used to amplify a polynucleotide isolated from aninfected individual, such that the polynucleotide may then be subject tovarious techniques for elucidation of the polynucleotide sequence. Inthis way, mutations in the polynucleotide sequence may be detected andused to diagnose and/or prognose the infection or its stage or course,or to serotype and/or classify the infectious agent.

The invention further provides a process for diagnosing, disease,preferably bacterial infections, more preferably infections caused bynon typeable H. influenzae, comprising determining from a sample derivedfrom an individual, such as a bodily material, an increased level ofexpression of polynucleotide having a sequence of any of the sequencesof SEQ Group 1. Increased or decreased expression of BASB231polynucleotide can be measured using any on of the methods well known inthe art for the quantitation of polynucleotides, such as, for example,amplification, PCR, RT-PCR, RNase protection, Northern blotting,spectrometry and other hybridization methods.

In addition, a diagnostic assay in accordance with the invention fordetecting over-expression of BASB231 polypeptide compared to normalcontrol tissue samples may be used to detect the presence of aninfection, for example. Assay techniques that can be used to determinelevels of BASB231 polypeptide, in a sample derived from a host, such asa bodily material, are well-known to those of skill in the art. Suchassay methods include radioimmunoassays, competitive-binding assays,Western Blot analysis, antibody sandwich assays, antibody detection andELISA assays.

The polynucleotides of the invention may be used as components ofpolynucleotide arrays, preferably high density arrays or grids. Thesehigh density arrays are particularly useful for diagnostic andprognostic purposes. For example, a set of spots each comprising adifferent gene, and further comprising a polynucleotide orpolynucleotides of the invention, may be used for probing, such as usinghybridization or nucleic acid amplification, using a probes obtained orderived from a bodily sample, to determine the presence of a particularpolynucleotide sequence or related sequence in an individual. Such apresence may indicate the presence of a pathogen, particularlynon-typeable Haemophilus influenzae, and may be useful in diagnosingand/or prognosing disease or a course of disease. A grid comprising anumber of variants of any polynucleotide sequence of SEQ Group 1 ispreferred. Also preferred is a number of variants of a polynucleotidesequence encoding any polypeptide sequence of SEQ Group 2.

Antibodies

The polypeptides and polynucleotides of the invention or variantsthereof, or cells expressing the same can be used as immunogens toproduce antibodies immunospecific for such polypeptides orpolynucleotides respectively. Alternatively, mimotopes, particularlypeptide mimotopes, of epitopes within the polypeptide sequence may alsobe used as immunogens to produce antibodies immunospecific for thepolypeptide of the invention. The term “immunospecific” means that theantibodies have substantially greater affinity for the polypeptides ofthe invention than their affinity for other related polypeptides in theprior art.

In certain preferred embodiments of the invention there are providedantibodies against BASB231 polypeptides or polynucleotides.

Antibodies generated against the polypeptides or polynucleotides of theinvention can be obtained by administering the polypeptides and/orpolynucleotides of the invention, or epitope-bearing fragments of eitheror both, analogues of either or both, or cells expressing either orboth, to an animal, preferably a nonhuman, using routine protocols. Forpreparation of monoclonal antibodies, any technique known in the artthat provides antibodies produced by continuous cell line cultures canbe used. Examples include various techniques, such as those in Kohler,G. and Milstein, C., Nature 256: 495497 (1975); Kozbor et al.,Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce single chain antibodies topolypeptides or polynucleotides of this invention. Also, transgenicmice, or other organisms or animals, such as other mammals, may be usedto express humanized antibodies immunospecific to the polypeptides orpolynucleotides of the invention.

Alternatively, phage display technology may be utilized to selectantibody genes with binding activities towards a polypeptide of theinvention either from repertoires of PCR amplified v-genes oflymphocytes from humans screened for possessing anti-BASB231 or fromnaive libraries (McCafferty, et al., (1990), Nature 348, 552-554; Marks,et al., (1992) Biotechnology 10, 779-783). The affinity of theseantibodies can also be improved by, for example, chain shuffling(Clackson et al., (1991) Nature 352: 628).

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptides or polynucleotides of the inventionto purify the polypeptides or polynucleotides by, for example, affinitychromatography.

Thus, among others, antibodies against BASB231 polypeptide or BASB231polynucleotide may be employed to treat infections, particularlybacterial infections.

Polypeptide variants include antigenically, epitopically orimmunologically equivalent variants form a particular aspect of thisinvention.

Preferably, the antibody or variant thereof is modified to make it lessimmunogenic in the individual. For example, if the individual is humanthe antibody may most preferably be “humanized,” where thecomplimentarily determining region or regions of the hybridoma-derivedantibody has been transplanted into a human monoclonal antibody, forexample as described in Jones et al. (1986), Nature 321, 522-525 orTempest et al., (1991) Biotechnology 9, 266-273.

Antagonists and Agonists—Assays and Molecules

Polypeptides and polynucleotides of the invention may also be used toassess the binding of small molecule substrates and ligands in, forexample, cells, cell-free preparations, chemical libraries, and naturalproduct mixtures. These substrates and ligands may be natural substratesand ligands or may be structural or functional mimetics. See, e.g.,Coligan et al., Current Protocols in Immunology 1(2): Chapter 5 (1991).

The screening methods may simply measure the binding of a candidatecompound to the polypeptide or polynucleotide, or to cells or membranesbearing the polypeptide or polynucleotide, or a fusion protein of thepolypeptide by means of a label directly or indirectly associated withthe candidate compound. Alternatively, the screening method may involvecompetition with a labeled competitor. Further, these screening methodsmay test whether the candidate compound results in a signal generated byactivation or inhibition of the polypeptide or polynucleotide, usingdetection systems appropriate to the cells comprising the polypeptide orpolynucleotide. Inhibitors of activation are generally assayed in thepresence of a known agonist and the effect on activation by the agonistby the presence of the candidate compound is observed. Constitutivelyactive polypeptide and/or constitutively expressed polypeptides andpolynucleotides may be employed in screening methods for inverseagonists or inhibitors, in the absence of an agonist or inhibitor, bytesting whether the candidate compound results in inhibition ofactivation of the polypeptide or polynucleotide, as the case may be.Further, the screening methods may simply comprise the steps of mixing acandidate compound with a solution containing a polypeptide orpolynucleotide of the present invention, to form a mixture, measuringBASB231 polypeptide and/or polynucleotide activity in the mixture, andcomparing the BASB231 polypeptide and/or polynucleotide activity of themixture to a standard. Fusion proteins, such as those made from Fcportion and BASB231 polypeptide, as hereinbefore described, can also beused for high-throughput screening assays to identify antagonists of thepolypeptide of the present invention, as well as of phylogenetically andand/or functionally related polypeptides (see D. Bennett et al., J MolRecognition, 8:52-58 (1995); and K. Johanson et al., J Biol Chem,270(16):9459-9471 (1995)).

The polynucleotides, polypeptides and antibodies that bind to and/orinteract with a polypeptide of the present invention may also be used toconfigure screening methods for detecting the effect of added compoundson the production of mRNA and/or polypeptide in cells. For example, anELISA assay may be constructed for measuring secreted or cell associatedlevels of polypeptide using monoclonal and polyclonal antibodies bystandard methods known in the art. This can be used to discover agentswhich may inhibit or enhance the production of polypeptide (also calledantagonist or agonist, respectively) from suitably manipulated cells ortissues.

The invention also provides a method of screening compounds to identifythose which enhance (agonist) or block (antagonist) the action ofBASB231 polypeptides or polynucleotides, particularly those compoundsthat are bacteriostatic and/or bactericidal. The method of screening mayinvolve high-throughput techniques. For example, to screen for agonistsor antagonists, a synthetic reaction mix, a cellular compartment, suchas a membrane, cell envelope or cell wall, or a preparation of anythereof, comprising BASB231 polypeptide and a labeled substrate orligand of such polypeptide is incubated in the absence or the presenceof a candidate molecule that may be a BASB231 agonist or antagonist. Theability of the candidate molecule to agonize or antagonize the BASB231polypeptide is reflected in decreased binding of the labeled ligand ordecreased production of product from such substrate. Molecules that bindgratuitously, i.e., without inducing the effects of BASB231 polypeptideare most likely to be good antagonists. Molecules that bind well and, asthe case may be, increase the rate of product production from substrate,increase signal transduction, or increase chemical channel activity areagonists. Detection of the rate or level of, as the case may be,production of product from substrate, signal transduction, or chemicalchannel activity may be enhanced by using a reporter system. Reportersystems that may be useful in this regard include but are not limited tocolorimetric, labeled substrate converted into product, a reporter genethat is responsive to changes in BASB231 polynucleotide or polypeptideactivity, and binding assays known in the art.

Another example of an assay for BASB231 agonists is a competitive assaythat combines BASB231 and a potential agonist with BASB231 bindingmolecules, recombinant BASB231 binding molecules, natural substrates orligands, or substrate or ligand mimetics, under appropriate conditionsfor a competitive inhibition assay. BASB231 can be labeled, such as byradioactivity or a colorimetric compound, such that the number ofBASB231 molecules bound to a binding molecule or converted to productcan be determined accurately to assess the effectiveness of thepotential antagonist.

Potential antagonists include, among others, small organic molecules,peptides, polypeptides and antibodies that bind to a polynucleotideand/or polypeptide of the invention and thereby inhibit or extinguishits activity or expression. Potential antagonists also may be smallorganic molecules, a peptide, a polypeptide such as a closely relatedprotein or antibody that binds the same sites on a binding molecule,such as a binding molecule, without inducing BASB231 induced activities,thereby preventing the action or expression of BASB231 polypeptidesand/or polynucleotides by excluding BASB231 polypeptides and/orpolynucleotides from binding.

Potential antagonists include a small molecule that binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall organic molecules, peptides or peptide-like molecules. Otherpotential antagonists include antisense molecules (see Okano, J.Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORSOF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for adescription of these molecules). Preferred potential antagonists includecompounds related to and variants of BASB231.

In a further aspect, the present invention relates to geneticallyengineered soluble fusion proteins comprising a polypeptide of thepresent invention, or a fragment thereof, and various portions of theconstant regions of heavy or light chains of immunoglobulins of varioussubclasses (IgG, IgM, IgA, IgE). Preferred as an immunoglobulin is theconstant part of the heavy chain of human IgG, particularly IgG1, wherefusion takes place at the hinge region. In a particular embodiment, theFc part can be removed simply by incorporation of a cleavage sequencewhich can be cleaved with blood clotting factor Xa. Furthermore, thisinvention relates to processes for the preparation of these fusionproteins by genetic engineering, and to the use thereof for drugscreening, diagnosis and therapy. A further aspect of the invention alsorelates to polynucleotides encoding such fusion proteins. Examples offusion protein technology can be found in International PatentApplication Nos. WO94/29458 and WO94/22914.

Each of the polynucleotide sequences provided herein may be used in thediscovery and development of antibacterial compounds. The encodedprotein, upon expression, can be used as a target for the screening ofantibacterial drugs. Additionally, the polynucleotide sequences encodingthe amino terminal regions of the encoded protein or Shine-Delgarno orother translation facilitating sequences of the respective mRNA can beused to construct antisense sequences to control the expression of thecoding sequence of interest.

The invention also provides the use of the polypeptide, polynucleotide,agonist or antagonist of the invention to interfere with the initialphysical interaction between a pathogen or pathogens and a eukaryotic,preferably mammalian, host responsible for sequelae of infection. Inparticular, the molecules of the invention may be used: in theprevention of adhesion of bacteria, in particular gram positive and/orgram negative bacteria, to eukaryotic, preferably mammalian,extracellular matrix proteins on in-dwelling devices or to extracellularmatrix proteins in wounds; to block bacterial adhesion betweeneukaryotic, preferably mammalian, extracellular matrix proteins andbacterial BASB231 proteins that mediate tissue damage and/or; to blockthe normal progression of pathogenesis in infections initiated otherthan by the implantation of in-dwelling devices or by other surgicaltechniques.

In accordance with yet another aspect of the invention, there areprovided BASB231 agonists and antagonists, preferably bacteristatic orbactericidal agonists and antagonists.

The antagonists and agonists of the invention may be employed, forinstance, to prevent, inhibit and/or treat diseases.

In a further aspect, the present invention relates to mimotopes of thepolypeptide of the invention. A mimotope is a peptide sequence,sufficiently similar to the native peptide (sequentially orstructurally), which is capable of being recognised by antibodies whichrecognise the native peptide; or is capable of raising antibodies whichrecognise the native peptide when coupled to a suitable carrier.

Peptide mimotopes may be designed for a particular purpose by addition,deletion or substitution of elected amino acids. Thus, the peptides maybe modified for the purposes of ease of conjugation to a proteincarrier. For example, it may be desirable for some chemical conjugationmethods to include a terminal cysteine. In addition it may be desirablefor peptides conjugated to a protein carrier to include a hydrophobicterminus distal from the conjugated terminus of the peptide, such thatthe free unconjugated end of the peptide remains associated with thesurface of the carrier protein. Thereby presenting the peptide in aconformation which most closely resembles that of the peptide as foundin the context of the whole native molecule. For example, the peptidesmay be altered to have an N-terminal cysteine and a C-terminalhydrophobic amidated tail. Alternatively, the addition or substitutionof a D-stereoisomer form of one or more of the amino acids may beperformed to create a beneficial derivative, for example to enhancestability of the peptide.

Alternatively, peptide mimotopes may be identified using antibodieswhich are capable themselves of binding to the polypeptides of thepresent invention using techniques such as phage display technology (EP0 552 267 B1). This technique, generates a large number of peptidesequences which mimic the structure of the native peptides and are,therefore, capable of binding to anti-native peptide antibodies, but maynot necessarily themselves share significant sequence homology to thenative polypeptide.

Vaccines

Another aspect of the invention relates to a method for inducing animmunological response in an individual, particularly a mammal,preferably humans, which comprises inoculating the individual withBASB231 polynucleotide and/or polypeptide, or a fragment or variantthereof, adequate to produce antibody and/or T cell immune response toprotect said individual from infection, particularly bacterial infectionand most particularly non typeable H. influenzae infection. Alsoprovided are methods whereby such immunological response slows bacterialreplication. Yet another aspect of the invention relates to a method ofinducing immunological response in an individual which comprisesdelivering to such individual a nucleic acid vector, sequence orribozyme to direct expression of BASB231 polynucleotide and/orpolypeptide, or a fragment or a variant thereof, for expressing BASB231polynucleotide and/or polypeptide, or a fragment or a variant thereof invivo in order to induce an immunological response, such as, to produceantibody and/or T cell immune response, including, for example,cytokine-producing T cells or cytotoxic T cells, to protect saidindividual, preferably a human, from disease, whether that disease isalready established within the individual or not. One example ofadministering the gene is by accelerating it into the desired cells as acoating on particles or otherwise. Such nucleic acid vector may compriseDNA, RNA, a ribozyme, a modified nucleic acid, a DNA/RNA hybrid, aDNA-protein complex or an RNA-protein complex.

A further aspect of the invention relates to an immunologicalcomposition that when introduced into an individual, preferably a human,capable of having induced within it an immunological response, inducesan immunological response in such individual to a BASB231 polynucleotideand/or polypeptide encoded therefrom, wherein the composition comprisesa recombinant BASB231 polynucleotide and/or polypeptide encodedtherefrom and/or comprises DNA and/or RNA which encodes and expresses anantigen of said BASB231 polynucleotide, polypeptide encoded therefrom,or other polypeptide of the invention. The immunological response may beused therapeutically or prophylactically and may take the form ofantibody immunity and/or cellular immunity, such as cellular immunityarising from CTL or CD4+ T cells.

BASB231 polypeptide or a fragment thereof may be fused with co-proteinor chemical moiety which may or may not by itself produce antibodies,but which is capable of stabilizing the first protein and producing afused or modified protein which will have antigenic and/or immunogenicproperties, and preferably protective properties. Thus fused recombinantprotein, preferably further comprises an antigenic co-protein, such aslipoprotein D from Haemophilus influenzae, Glutathione-S-transferase(GST) or beta-galactosidase, or any other relatively large co-proteinwhich solubilizes the protein and facilitates production andpurification thereof. Moreover, the co-protein may act as an adjuvant inthe sense of providing a generalized stimulation of the immune system ofthe organism receiving the protein. The co-protein may be attached toeither the amino- or carboxy-terminus of the first protein.

In a vaccine composition according to the invention, a BASB231polypeptide and/or polynucleotide, or a fragment, or a mimotope, or avariant thereof may be present in a vector, such as the live recombinantvectors described above for example live bacterial vectors.

Also suitable are non-live vectors for the BASB231 polypeptide, forexample bacterial outer-membrane vesicles or “blebs”. OM blebs arederived from the outer membrane of the two-layer membrane ofGram-negative bacteria and have been documented in many Gram-negativebacteria (Zhou, L et al. 1998. FEMS Microbiol. Lett. 163:223-228)including C. trachomatis and C. psittaci. A non-exhaustive list ofbacterial pathogens reported to produce blebs also includes: Bordetellapertussis, Borrelia burgdorferi, Brucella melitensis, Brucella ovis,Esherichia coli, Haemophilus influenzae, Legionella pneumophila,Moraxella catarrhalis, Neisseria gonorrhoeae, Neisseria meningitidis,Pseudomonas aeruginosa and Yersinia enterocolitica.

Blebs have the advantage of providing outer-membrane proteins in theirnative conformation and are thus particularly useful for vaccines. Blebscan also be improved for vaccine use by engineering the bacterium so asto modify the expression of one or more molecules at the outer membrane.Thus for example the expression of a desired immunogenic protein at theouter membrane, such as the BASB231 polypeptide, can be introduced orupregulated (e.g. by altering the promoter). Instead or in addition, theexpression of outer-membrane molecules which are either not relevant(e.g. unprotective antigens or immunodominant but variable proteins) ordetrimental (e.g. toxic molecules such as LPS, or potential inducers ofan autoimmune response) can be down-regulated. These approaches arediscussed in more detail below.

The non-coding flanking regions of the BASB231 gene contain regulatoryelements important in the expression of the gene. This regulation takesplace both at the transcriptional and translational level. The sequenceof these regions, either upstream or downstream of the open readingframe of the gene, can be obtained by DNA sequencing. This sequenceinformation allows the determination of potential regulatory motifs suchas the different promoter elements, terminator sequences, induciblesequence elements, repressors, elements responsible for phase variation,the shine-dalgarno sequence, regions with potential secondary structureinvolved in regulation, as well as other types of regulatory motifs orsequences. This sequence is a further aspect of the invention.Furthermore, SEQ ID NO: 75 contains the non typeable Haemophilusinfluenzae polynucleotide sequences not present in the HiRd genome andcomprising the ORFs1, 2, 3, 4, 5, 6, 7, 8 and their non-coding flankingregions.

The non-coding flanking regions are located between the ORFs of SED IDNO: 75. The localisation of the ORFs of SED ID NO: 75 are listed intable 4. TABLE 4 Position of the Position of the first nucleotide oflast nucleotide of stop Name start codon codon Strand Orf1  90 542 +Orf2  545 1576 + Orf3 2391 1579 − Orf4 3165 2440 − Orf5 3915 3175 − Orf64934 3912 − Orf7 5881 4940 − Orf6  6579* 6022 −*It is not the start codon, it is the first nucleotide of the codingsequence

Furthermore, SEQ ID NO: 76 contains the non typeable Haemophilusinfluenzae polynucleotide sequences not present in the HiRd genome andcomprising the ORFs 9, 10, 11, 12, 13 and their non-coding flankingregions.

The non-coding flanking regions are located between the ORFs of SED IDNO: 76. The localisation of the ORFs of SED ID NO: 76 are listed intable 5. TABLE 5 Position of the Position of the last first nucleotideof nucleotide of stop Name start codon codon Strand Orf9 140 2512 +Orf10 2695 3512 + Orf11 3470 4104 + Orf12 4270 5526 + Orf13 5626 8652 +

Furthermore, SEQ ID NO: 77 contains the non typeable Haemophilusinfluenzae polynucleotide sequences not present in the HiRd genome andcomprising the ORFs 14, 15, 16, 17, 18, 19, 20, 21, 22 and theirnon-coding flanking regions.

The non-coding flanking regions are located between the ORFs of SED IDNO: 77. The localisation of the ORFs of SED ID NO: 77 are listed intable 6. TABLE 6 Position of the Position of the last first nucleotideof nucleotide of stop Name start codon codon Strand Orf14 2110 54 −Orf15 3161 2187 − Orf16  3931* 3239 − Orf17 4854 4039 − Orf18  5123*4851 − Orf19 5246 6268 + Orf20 7027 6317 − Orf21 7467 7011 − Orf22 7966* 7526 −*It is not the first nucleotide of the strat codon, it is the firstnucleotide of the coding sequence

Furthermore, SEQ ID) NO: 78 contains the non typeable Haemophilusinfluenzae polynucleotide sequences not present in the HiRd genome andcomprising the ORFs 23, 24 and their non-coding flanking regions.

The non-coding flanking regions are located between the ORFs of SED IDNO: 78. The localisation of the ORFs of SED ID NO: 78 are listed intable 7. TABLE 7 Position of the Position of the last first nucleotideof nucleotide of stop Name start codon codon Strand Orf23 688 47 − Orf242028 685 −

Furthermore, SEQ ID NO: 79 contains the non typeable Haemophilusinfluenzae polynucleotide sequences not present in the HiRd genome andcomprising the ORF 25 and their non-coding flanking regions.

The non-coding flanking regions are located between the ORF of SED IDNO: 79. The localisation of the ORF of SED ID NO: 79 are listed in table8. TABLE 8 Position of the Position of the first nucleotide of lastnucleotide of stop Name start codon codon Strand Orf25 2205 211 −

Furthermore, SEQ ID NO: 80 contains the non typeable Haemophilusinfluenzae polynucleotide sequences not present in the HiRd genome andcomprising the ORFs 26, 27 and their non-coding flanking regions.

The non-coding flanking regions are located between the ORFs of SED IDNO: 80. The localisation of the ORFs of SED ID NO: 80 are listed intable 9. TABLE 9 Position of the Position of the first nucleotide oflast nucleotide of stop Name start codon codon Strand Orf26   34* 1182 +Orf27 1187 2185 +*It is not the first nucleotide of the strat codon, it is the firstnucleotide of the coding sequence

Furthermore, SEQ ID NO: 81 contains the non typeable Haemophilusinfluenzae polynucleotide sequences not present in the HiRd genome andcomprising the ORFs 28, 29 and their non-coding flanking regions.

The non-coding flanking regions are located between the ORFs of SED IDNO: 81. The localisation of the ORFs of SED ID NO: 81 are listed intable 10. TABLE 10 Position of the Position of the first nucleotide oflast nucleotide of stop Name start codon codon Strand Orf28 152 970 +Orf29 1729* 1397 −*It is not the first nucleotide of the strat codon, it is the firstnucleotide of the coding sequence

Furthermore, SEQ ID NO: 82 contains the non typeable Haemophilusinfluenzae polynucleotide sequences not present in the HiRd genome andcomprising the ORFs 30, 31, 32 and their non-coding flanking regions.

The non-coding flanking regions are located between the ORFs of SED IDNO: 82. The localisation of the ORFs of SED ID NO: 82 are listed intable 11. TABLE 11 Position of the Position of the first nucleotide oflast nucleotide of stop Name start codon codon Strand Orf30  271 11 −Orf31 1154 237 − Orf32  1475* 1164 −*It is not the first nucleotide of the strat codon, it is the firstnucleotide of the coding sequence

Furthermore, SEQ ID NO: 83 contains the non typeable Haemophilusinfluenzae polynucleotide sequences not present in the HiRd genome andcomprising the ORE 33 and their non-coding flanking regions.

The non-coding flanking regions are located between the ORF of SED IDNO: 83. The localisation of the ORF of SED ID NO: 83 are listed in table12. TABLE 12 Position of the Position of the first nucleotide of lastnucleotide of stop Name start codon codon Strand Orf33 74 1537 +

Furthermore, SEQ ID NO: 84 contains the non typeable Haemophilusinfluenzae polynucleotide sequences not present in the HiRd genome andcomprising the ORF 34 and their non-coding flanking regions.

The non-coding flanking regions are located between the ORF of SED IDNO: 84. The localisation of the ORF of SED ID NO: 84 are listed in table13. TABLE 13 Position of the Position of the first nucleotide of lastnucleotide of stop Name start codon codon Strand Orf34 82 969 +

Furthermore, SEQ ID NO: 85 contains the non typeable Haemophilusinfluenzae polynucleotide sequences not present in the HiRd genome andcomprising the ORF 35 and their non-coding flanking regions.

The non-coding flanking regions are located between the ORF of SED IDNO: 83. The localisation of the ORF of SED ID NO: 85 are listed in table13. TABLE 13 Position of the Position of the first nucleotide of lastnucleotide of stop Name start codon codon Strand Orf35 1065* 223 −*It is not the first nucleotide of the strat codon, it is the firstnucleotide of the coding sequence

Furthermore, SEQ ID NO: 86 contains the non typeable Haemophilusinfluenzae polynucleotide sequences not present in the HiRd genome andcomprising the ORF 36 and their non-coding flanking regions.

The non-coding flanking regions are located between the ORF of SED IDNO: 86. The localisation of the ORF of SED ID NO: 86 are listed in table14. TABLE 14 Position of the Position of the first nucleotide of lastnucleotide of stop Name start codon codon Strand Orf36 254* 646 +*It is not the first nucleotide of the strat codon, it is the firstnucleotide of the coding sequence

Furthermore, SEQ ID NO: 87 contains the non typeable Haemophilusinfluenzae polynucleotide sequences not present in the HiRd genome andcomprising the ORF 37 and their non-coding flanking regions.

The non-coding flanking regions are located between the ORF of SED IDNO: 87. The localisation of the ORF of SED ID NO: 87 are listed in table15. TABLE 15 Position of the Position of the first nucleotide of lastnucleotide of stop Name start codon codon Strand Orf37 202* 876 +

This sequence information allows the modulation of the naturalexpression of the BASB231 gene. The upregulation of the gene expressionmay be accomplished by altering the promoter, the shine-dalgarnosequence, potential repressor or operator elements, or any otherelements involved. Likewise, downregulation of expression can beachieved by similar types of modification. Alternatively, by changingphase variation sequences, the expression of the gene can be put underphase variation control, or it may be uncoupled from this regulation. Inanother approach, the expression of the gene can be put under thecontrol of one or more inducible elements allowing regulated expression.Examples of such regulation include, but are not limited to, inductionby temperature shift, addition of inductor substrates like selectedcarbohydrates or their derivatives, trace elements, vitamins,co-factors, metal ions, etc.

Such modifications as described above can be introduced by severaldifferent means. The modification of sequences involved in geneexpression can be carried out in vivo by random mutagenesis followed byselection for the desired phenotype. Another approach consists inisolating the region of interest and modifying it by random mutagenesis,or site-directed replacement, insertion or deletion mutagenesis. Themodified region can then be reintroduced into the bacterial genome byhomologous recombination, and the effect on gene expression can beassessed. In another approach, the sequence knowledge of the region ofinterest can be used to replace or delete all or part of the naturalregulatory sequences. In this case, the regulatory region targeted isisolated and modified so as to contain the regulatory elements fromanother gene, a combination of regulatory elements from different genes,a synthetic regulatory region, or any other regulatory region, or todelete selected parts of the wild-type regulatory sequences. Thesemodified sequences can then be reintroduced into the bacterium viahomologous recombination into the genome. A non-exhaustive list ofpreferred promoters that could be used for up-regulation of geneexpression includes the promoters porA, porB, lbpB, tbpB, p110, 1st,hpuAB from N. meningitidis or N. gonorroheae; ompCD, copB, lbpB, ompE,UspA1; UspA2; TbpB from M. Catarrhalis; p1, p2, p4, p5, p6, IpD, tbpB,D15, Hia, Hmw1, Hmw2 from H. influenzae.

In one example, the expression of the gene can be modulated byexchanging its promoter with a stronger promoter (through isolating theupstream sequence of the gene, in vitro modification of this sequence,and reintroduction into the genome by homologous recombination).Upregulated expression can be obtained in both the bacterium as well asin the outer membrane vesicles shed (or made) from the bacterium.

In other examples, the described approaches can be used to generaterecombinant bacterial strains with improved characteristics for vaccineapplications. These can be, but are not limited to, attenuated strains,strains with increased expression of selected antigens, strains withknock-outs (or decreased expression) of genes interfering with theimmune response, strains with modulated expression of immunodominantproteins, strains with modulated shedding of outer-membrane vesicles.

Thus, also provided by the invention is a modified upstream region ofthe BASB231 gene, which modified upstream region contains a heterologousregulatory element which alters the expression level of the BASB231protein located at the outer membrane. The upstream region according tothis aspect of the invention includes the sequence upstream of theBASB231 gene. The upstream region starts immediately upstream of theBASB231 gene and continues usually to a position no more than about 1000bp upstream of the gene from the ATG start codon. In the case of a genelocated in a polycistronic sequence (operon) the upstream region canstart immediately preceding the gene of interest, or preceding the firstgene in the operon. Preferably, a modified upstream region according tothis aspect of the invention contains a heterologous promotor at aposition between 500 and 700 bp upstream of the ATG.

The use of the disclosed upstream regions to upregulate the expressionof the BASB231 gene, a process for achieving this through homologousrecombination (for instance as described in WO 01/09350 incorporated byreference herein), a vector comprising upstream sequence suitable forthis purpose, and a host cell so altered are all further aspects of thisinvention.

Thus, the invention provides a BASB231 polypeptide, in a modifiedbacterial bleb. The invention further provides modified host cellscapable of producing the non-live membrane-based bleb vectors. Theinvention further provides nucleic acid vectors comprising the BASB231gene having a modified upstream region containing a heterologousregulatory element.

Further provided by the invention are processes to prepare the hostcells and bacterial blebs according to the invention.

Also provided by this invention are compositions, particularly vaccinecompositions, and methods comprising the polypeptides and/orpolynucleotides of the invention and immunostimulatory DNA sequences,such as those described in Sato, Y. et al. Science 273: 352 (1996).

Also, provided by this invention are methods using the describedpolynucleotide or particular fragments thereof, which have been shown toencode non-variable regions of bacterial cell surface proteins, inpolynucleotide constructs used in such genetic immunization experimentsin animal models of infection with non typeable H. influenzae. Suchexperiments will be particularly useful for identifying protein epitopesable to provoke a prophylactic or therapeutic immune response. It isbelieved that this approach will allow for the subsequent preparation ofmonoclonal antibodies of particular value, derived from the requisiteorgan of the animal successfully resisting or clearing infection, forthe development of prophylactic agents or therapeutic treatments ofbacterial infection, particularly non typeable H. influenzae infection,in mammals, particularly humans.

The invention also includes a vaccine formulation which comprises animmunogenic recombinant polypeptide and/or polynucleotide of theinvention together with a suitable carrier, such as a pharmaceuticallyacceptable carrier. Since the polypeptides and polynucleotides may bebroken down in the stomach, each is preferably administeredparenterally, including, for example, administration that issubcutaneous, intramuscular, intravenous, or intradermal. Formulationssuitable for parenteral administration include aqueous and non-aqueoussterile injection solutions which may contain anti-oxidants, buffers,bacteriostatic compounds and solutes which render the formulationisotonic with the bodily fluid, preferably the blood, of the individual;and aqueous and non-aqueous sterile suspensions which may includesuspending agents or thickening agents. The formulations may bepresented in unit-dose or multi-dose containers, for example, sealedampoules and vials and may be stored in a freeze-dried conditionrequiring only the addition of the sterile liquid carrier immediatelyprior to use.

The vaccine formulation of the invention may also include adjuvantsystems for enhancing the immunogenicity of the formulation. Preferablythe adjuvant system raises preferentially a TH1 type of response.

An immune response may be broadly distinguished into two extremecatagories, being a humoral or cell mediated immune responses(traditionally characterised by antibody and cellular effectormechanisms of protection respectively). These categories of responsehave been termed TH1-type responses (cell-mediated response), andTH2-type immune responses (humoral response).

Extreme TH1-type immune responses may be characterised by the generationof antigen specific, haplotype restricted cytotoxic T lymphocytes, andnatural killer cell responses. In mice TH 1-type responses are oftencharacterised by the generation of antibodies of the IgG2a subtype,whilst in the human these correspond to IgG1 type antibodies. TH2-typeimmune responses are characterised by the generation of a broad range ofimmunoglobulin isotypes including in mice IgG1, IgA, and IgM.

It can be considered that the driving force behind the development ofthese two types of immune responses are cytokines. High levels ofTH1-type cytokines tend to favour the induction of cell mediated immuneresponses to the given antigen, whilst high levels of TH2-type cytokinestend to favour the induction of humoral immune responses to the antigen.

The distinction of TH1 and TH2-type immune responses is not absolute. Inreality an individual will support an immune response which is describedas being predominantly TH1 or predominantly TH2. However, it is oftenconvenient to consider the families of cytokines in terms of thatdescribed in murine CD4+ ve T cell clones by Mosmann and Coffman(Mosmann, T R. and Coffman, R. L. (1989) TH1 and TH2 cells: differentpatterns of lymphokine secretion lead to different functionalproperties. Annual Review of Immunology, 7, p145-173). Traditionally,TH1-type responses are associated with the production of the INF-γ andIL-2 cytokines by T-lymphocytes. Other cytokines often directlyassociated with the induction of TH1-type immune responses are notproduced by T-cells, such as IL-12. In contrast, TH2-type responses areassociated with the secretion of IL-4, IL-5, IL-6 and IL-13.

It is known that certain vaccine adjuvants are particularly suited tothe stimulation of either TH1 or TH2-type cytokine responses.Traditionally the best indicators of the TH1:TH2 balance of the immuneresponse after a vaccination or infection includes direct measurement ofthe production of TH1 or TH2 cytokines by T lymphocytes in vitro afterrestimulation with antigen, and/or the measurement of the IgG1:IgG2aratio of antigen specific antibody responses.

Thus, a TH1-type adjuvant is one which preferentially stimulatesisolated T-cell populations to produce high levels of TH1-type cytokineswhen re-stimulated with antigen in vitro, and promotes development ofboth CD8+ cytotoxic T lymphocytes and antigen specific immunoglobulinresponses associated with TH1-type isotype.

Adjuvants which are capable of preferential stimulation of the TH1 cellresponse are described in International Patent Application No. WO94/00153 and WO 95/17209.

3 De-O-acylated monophosphoryl lipid A (3D-MPL) is one such adjuvant.This is known from GB 2220211 (Ribi). Chemically it is a mixture of 3De-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains andis manufactured by Ribi Immunochem, Montana. A preferred form of 3De-O-acylated monophosphoryl lipid A is disclosed in European Patent 0689 454 B1 (SmithKline Beecham Biologicals SA).

Preferably, the particles of 3D-MPL are small enough to be sterilefiltered through a 0.22 micron membrane (European Patent number 0 689454).

3D-MPL will be present in the range of 10 μg-100 μg preferably 25-50 μgper dose wherein the antigen will typically be present in a range 2-50μg per dose.

Another preferred adjuvant comprises QS21, an Hplc purified non-toxicfraction derived from the bark of Quillaja Saponaria Molina. Optionallythis may be admixed with 3 De-O-acylated monophosphoryl lipid A(3D-MPL), optionally together with an carrier.

The method of production of QS21 is disclosed in U.S. Pat. No.5,057,540.

Non-reactogenic adjuvant formulations containing QS21 have beendescribed previously (WO 96/33739). Such formulations comprising QS21and cholesterol have been shown to be successful TH1 stimulatingadjuvants when formulated together with an antigen.

Further adjuvants which are preferential stimulators of TH1 cellresponse include immunomodulatory oligonucleotides, for exampleunmethylated CpG sequences as disclosed in WO 96/02555.

Combinations of different TH1 stimulating adjuvants, such as thosementioned hereinabove, are also contemplated as providing an adjuvantwhich is a preferential stimulator of TH1 cell response. For example,QS21 can be formulated together with 3D-MPL. The ratio of QS21:3D-MPLwill typically be in the order of 1:10 to 10:1; preferably 1:5 to 5:1and often substantially 1:1. The preferred range for optimal synergy is2.5:1 to 1:1 3D-MPL: QS21.

Preferably a carrier is also present in the vaccine compositionaccording to the invention. The carrier may be an oil in water emulsion,or an aluminium salt, such as aluminium phosphate or aluminiumhydroxide.

A preferred oil-in-water emulsion comprises a metabolisible oil, such assqualene, alpha tocopherol and Tween 80. In a particularly preferredaspect the antigens in the vaccine composition according to theinvention are combined with QS21 and 3D-MPL in such an emulsion.Additionally the oil in water emulsion may contain span 85 and/orlecithin and/or tricaprylin.

Typically for human administration QS21 and 3D-MPL will be present in avaccine in the range of 1 μg-200 μg, such as 10-100 μg, preferably 10μg-50 μg per dose. Typically the oil in water will comprise from 2 to10% squalene, from 2 to 10% alpha tocopherol and from 0.3 to 3% tween80. Preferably the ratio of squalene: alpha tocopherol is equal to orless than 1 as this provides a more stable emulsion. Span 85 may also bepresent at a level of 1%. In some cases it may be advantageous that thevaccines of the present invention will further contain a stabiliser.

Non-toxic oil in water emulsions preferably contain a non-toxic oil,e.g. squalane or squalene, an emulsifier, e.g. Tween 80, in an aqueouscarrier. The aqueous carrier may be, for example, phosphate bufferedsaline.

A particularly potent adjuvant formulation involving QS21, 3D-MPL andtocopherol in an oil in water emulsion is described in WO 95/17210.

While the invention has been described with reference to certain BASB231polypeptides and polynucleotides, it is to be understood that thiscovers fragments of the naturally occurring polypeptides andpolynucleotides, and similar polypeptides and polynucleotides withadditions, deletions or substitutions which do not substantially affectthe immunogenic properties of the recombinant polypeptides orpolynucleotides.

The present invention also provides a polyvalent vaccine compositioncomprising a vaccine formulation of the invention in combination withother antigens, in particular antigens useful for treating otitis media.Such a polyvalent vaccine composition may include a TH-1 inducingadjuvant as hereinbefore described.

In a preferred embodiment, the polypeptides, fragments and immunogens ofthe invention are formulated with one or more of the following groups ofantigens: a) one or more pneumococcal capsular polysaccharides (eitherplain or conjugated to a carrier protein); b) one or more antigens thatcan protect a host against M. catarrhalis infection; c) one or moreprotein antigens that can protect a host against Streptococcuspneumoniae infection; d) one or more further non typeable Haemophilusinfluenzae protein antigens; e) one or more antigens that can protect ahost against RSV; and f) one or more antigens that can protect a hostagainst influenza virus. Combinations with: groups a) and b); b) and c);b), d), and a) and/or c); b), d), e), f), and a) and/or c) arepreferred. Such vaccines may be advantageously used as global otitismedia vaccines.

The pneumococcal capsular polysaccharide antigens are preferablyselected from serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F,14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F (most preferably fromserotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F).

Preferred pneumococcal protein antigens are those pneumococcal proteinswhich are exposed on the outer surface of the pneumococcus (capable ofbeing recognised by a host's immune system during at least part of thelife cycle of the pneumococcus), or are proteins which are secreted orreleased by the pneumococcus. Most preferably, the protein is a toxin,adhesin, 2-component signal tranducer, or lipoprotein of Streptococcuspneumoniae, or fragments thereof. Particularly preferred proteinsinclude, but are not limited to: pneurnolysin (preferably detoxified bychemical treatment or mutation) [Mitchell et al. Nucleic Acids Res. 1990Jul. 11; 18(13): 4010 “Comparison of pneumolysin genes and proteins fromStreptococcus pneumoniae types 1 and 2.”, Mitchell et al. BiochimBiophys Acta 1989 Jan. 23; 1007(1): 67-72 “Expression of the pneumolysingene in Escherichia coli: rapid purification and biologicalproperties.”, WO 96/05859 (A. Cyanamid), WO 90/06951 (Paton et al), WO99/03884 (NAVA)]; PspA and transmembrane deletion variants thereof (U.S.Pat. No. 5,804,193—Briles et al.); PspC and transmembrane deletionvariants thereof (WO 97/09994—Briles et al); PsaA and transmembranedeletion variants thereof (Berry & Paton, Infect Immun 1996December;64(12):5255-62 “Sequence heterogeneity of PsaA, a 37-kilodaltonputative adhesin essential for virulence of Streptococcus pneumoniae”);pneumococcal choline binding proteins and transmembrane deletionvariants thereof; CbpA and transmembrane deletion variants thereof (WO97/41151; WO 99/51266); Glyceraldehyde-3-phosphate—dehydrogenase(Infect. Immun. 1996 64:3544); HSP70 (WO 96/40928); PcpA (Sanchez-Beatoet al. FEMS Microbiol Lett 1998, 164:207-14); M like protein, SB patentapplication No. EP 0837130; and adhesin 18627, SB Patent application No.EP 0834568. Further preferred pneumococcal protein antigens are thosedisclosed in WO 98/18931, particularly those selected in WO 98/18930 andPCT/US99/30390.

Preferred further non-typeable H. influenzae protein antigens includeFimbrin protein (U.S. Pat. No. 5,766,608) and fusions comprisingpeptides therefrom (eg LB1 Fusion) (U.S. Pat. No. 5,843,464—Ohio StateResearch Foundation), OMP26, P6, protein D, ThpA, TbpB, Hia, Hmw1, Hmw2,Hap, and D15.

Preferred influenza virus antigens include whole, live or inactivatedvirus, split influenza virus, grown in eggs or MDCK cells, or Vero cellsor whole flu virosomes (as described by R. Gluck, Vaccine, 1992, 10,915-920) or purified or recombinant proteins thereof, such as HA, NP,NA, or M proteins, or combinations thereof.

Preferred RSV (Respiratory Syncytial Virus) antigens include the Fglycoprotein, the G glycoprotein, the HN protein, or derivativesthereof.

Compositions, Kits and Administration

In a further aspect of the invention there are provided compositionscomprising a BASB231 polynucleotide and/or a BASB231 polypeptide foradministration to a cell or to a multicellular organism.

The invention also relates to compositions comprising a polynucleotideand/or a polypeptides discussed herein or their agonists or antagonists.The polypeptides and polynucleotides of the invention may be employed incombination with a non-sterile or sterile carrier or carriers for usewith cells, tissues or organisms, such as a pharmaceutical carriersuitable for administration to an individual. Such compositionscomprise, for instance, a media additive or a therapeutically effectiveamount of a polypeptide and/or polynucleotide of the invention and apharmaceutically acceptable carrier or excipient. Such carriers mayinclude, but are not limited to, saline, buffered saline, dextrose,water, glycerol, ethanol and combinations thereof. The formulationshould suit the mode of administration. The invention further relates todiagnostic and pharmaceutical packs and kits comprising one or morecontainers filled with one or more of the ingredients of theaforementioned compositions of the invention.

Polypeptides, polynucleotides and other compounds of the invention maybe employed alone or in conjunction with other compounds, such astherapeutic compounds.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes among others.

In therapy or as a prophylactic, the active agent may be administered toan individual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

In a further aspect, the present invention provides for pharmaceuticalcompositions comprising a therapeutically effective amount of apolypeptide and/or polynucleotide, such as the soluble form of apolypeptide and/or polynucleotide of the present invention, agonist orantagonist peptide or small molecule compound, in combination with apharmaceutically acceptable carrier or excipient. Such carriers include,but are not limited to, saline, buffered saline, dextrose, water,glycerol, ethanol, and combinations thereof. The invention furtherrelates to pharmaceutical packs and kits comprising one or morecontainers filled with one or more of the ingredients of theaforementioned compositions of the invention. Polypeptides,polynucleotides and other compounds of the present invention may beemployed alone or in conjunction with other compounds, such astherapeutic compounds.

The composition will be adapted to the route of administration, forinstance by a systemic or an oral route. Preferred forms of systemicadministration include injection, typically by intravenous injection.Other injection routes, such as subcutaneous, intramuscular, orintraperitoneal, can be used. Alternative means for systemicadministration include transmucosal and transdermal administration usingpenetrants such as bile salts or fusidic acids or other detergents. Inaddition, if a polypeptide or other compounds of the present inventioncan be formulated in an enteric or an encapsulated formulation, oraladministration may also be possible. Administration of these compoundsmay also be topical and/or localized, in the form of salves, pastes,gels, solutions, powders and the like.

For administration to mammals, and particularly humans, it is expectedthat the daily dosage level of the active agent will be from 0.01 mg/kgto 10 mg/kg, typically around 1 mg/kg. The physician in any event willdetermine the actual dosage which will be most suitable for anindividual and will vary with the age, weight and response of theparticular individual. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of thisinvention.

The dosage range required depends on the choice of peptide, the route ofadministration, the nature of the formulation, the nature of thesubject's condition, and the judgment of the attending practitioner.Suitable dosages, however, are in the range of 0.1-100 μg/kg of subject.

A vaccine composition is conveniently in injectable form. Conventionaladjuvants may be employed to enhance the immune response. A suitableunit dose for vaccination is 0.5-5 microgram/kg of antigen, and suchdose is preferably administered 1-3 times and with an interval of 1-3weeks. With the indicated dose range, no adverse toxicological effectswill be observed with the compounds of the invention which wouldpreclude their administration to suitable individuals.

Wide variations in the needed dosage, however, are to be expected inview of the variety of compounds available and the differingefficiencies of various routes of administration. For example, oraladministration would be expected to require higher dosages thanadministration by intravenous injection. Variations in these dosagelevels can be adjusted using standard empirical routines foroptimization, as is well understood in the art.

Sequence Databases, Sequences in a Tangible Medium, and Algorithms

Polynucleotide and polypeptide sequences form a valuable informationresource with which to determine their 2- and 3-dimensional structuresas well as to identify further sequences of similar homology. Theseapproaches are most easily facilitated by storing the sequence in acomputer readable medium and then using the stored data in a knownmacromolecular structure program or to search a sequence database usingwell known searching tools, such as the GCG program package.

Also provided by the invention are methods for the analysis of charactersequences or strings, particularly genetic sequences or encoded proteinsequences. Preferred methods of sequence analysis include, for example,methods of sequence homology analysis, such as identity and similarityanalysis, DNA, RNA and protein structure analysis, sequence assembly,cladistic analysis, sequence motif analysis, open reading framedetermination, nucleic acid base calling, codon usage analysis, nucleicacid base trimming, and sequencing chromatogram peak analysis.

A computer based method is provided for performing homologyidentification. This method comprises the steps of: providing a firstpolynucleotide sequence comprising the sequence of a polynucleotide ofthe invention in a computer readable medium; and comparing said firstpolynucleotide sequence to at least one second polynucleotide orpolypeptide sequence to identify homology.

A computer based method is also provided for performing homologyidentification, said method comprising the steps of: providing a firstpolypeptide sequence comprising the sequence of a polypeptide of theinvention in a computer readable medium; and comparing said firstpolypeptide sequence to at least one second polynucleotide orpolypeptide sequence to identify homology.

All publications and references, including but not limited to patentsand patent applications, cited in this specification are hereinincorporated by reference in their entirety as if each individualpublication or reference were specifically and individually indicated tobe incorporated by reference herein as being fully set forth. Any patentapplication to which this application claims priority is alsoincorporated by reference herein in its entirety in the manner describedabove for publications and references.

Definitions

“Identity,” as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, as thecase may be, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”can be readily calculated by known methods, including but not limited tothose described in (Computational Molecular Biology, Lesk, A. M., ed.,Oxford University Press, New York, 1988; Biocomputing: Informatics andGenome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heine, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math.,48: 1073 (1988). Methods to determine identity are designed to give thelargest match between the sequences tested. Moreover, methods todetermine identity are codified in publicly available computer programs.Computer program methods to determine identity between two sequencesinclude, but are not limited to, the GAP program in the GCG programpackage (Devereux, J., et al., Nucleic Acids Research 12(1): 387(1984)), BLASTP, BLASTN (Altschul, S. F. et al., J. Molec. Biol. 215:403-410 (1990), and FASTA (Pearson and Lipman Proc. Natl. Acad. Sci. USA85; 2444-2448 (1988). The BLAST family of programs is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). The well known Smith Waterman algorithm may also be usedto determine identity.

Parameters for polypeptide sequence comparison include the following:

-   Algorithm: Needleman and Wunsch, J. Mol. Biol. 48: 443-453 (1970)-   Comparison matrix: BLOSSUM62 from Henikoff and Henikoff,-   Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992)-   Gap Penalty: 8-   Gap Length Penalty: 2

A program useful with these parameters is publicly available as the“gap” program from Genetics Computer Group, Madison Wis. Theaforementioned parameters are the default parameters for peptidecomparisons (along with no penalty for end gaps).

Parameters for polynucleotide comparison include the following:

-   Algorithm: Needleman and Wunsch, J. Mol. Biol. 48: 443-453 (1970)-   Comparison matrix: matches=+10, mismatch=0-   Gap Penalty: 50-   Gap Length Penalty: 3-   Available as: The “gap” program from Genetics Computer Group,    Madison Wis. These are the default parameters for nucleic acid    comparisons.

A preferred meaning for “identity” for polynucleotides and polypeptides,as the case may be, are provided in (1) and (2) below.

(1) Polynucleotide embodiments further include an isolatedpolynucleotide comprising a polynucleotide sequence having at least a50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the referencesequence of SEQ ID NO:1, wherein said polynucleotide sequence may beidentical to the reference sequence of SEQ ID NO:1 or may include up toa certain integer number of nucleotide alterations as compared to thereference sequence, wherein said alterations are selected from the groupconsisting of at least one nucleotide deletion, substitution, includingtransition and transversion, or insertion, and wherein said alterationsmay occur at the 5′ or 3′ terminal positions of the reference nucleotidesequence or anywhere between those terminal positions, interspersedeither individually among the nucleotides in the reference sequence orin one or more contiguous groups within the reference sequence, andwherein said number of nucleotide alterations is determined bymultiplying the total number of nucleotides in SEQ ID NO:1 by theinteger defining the percent identity divided by 100 and thensubtracting that product from said total number of nucleotides in SEQ IDNO:1, or:n _(n)≦x_(n)−(x _(n) ·y),wherein n_(n) is the number of nucleotide alterations, x_(n) is thetotal number of nucleotides in SEQ ID NO:1, y is 0.50 for 50%, 0.60 for60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for95%, 0.97 for 97% or 1.00 for 100%, and · is the symbol for themultiplication operator, and wherein any non-integer product of x_(n)and y is rounded down to the nearest integer prior to subtracting itfrom x_(n). Alterations of polynucleotide sequences encoding thepolypeptides of SEQ ID NO:2 may create nonsense, missense or frameshiftmutations in this coding sequence and thereby alter the polypeptideencoded by the polynucleotide following such alterations.

By way of example, a polynucleotide sequence of the present inventionmay be identical to the reference sequences of SEQ ID NO:1, that is itmay be 100% identical, or it may include up to a certain integer numberof nucleic acid alterations as compared to the reference sequence suchthat the percent identity is less than 100% identity. Such alterationsare selected from the group consisting of at least one nucleic aciddeletion, substitution, including transition and transversion, orinsertion, and wherein said alterations may occur at the 5′ or 3′terminal positions of the reference polynucleotide sequence or anywherebetween those terminal positions, interspersed either individually amongthe nucleic acids in the reference sequence or in one or more contiguousgroups within the reference sequence. The number of nucleic acidalterations for a given percent identity is determined by multiplyingthe total number of nucleic acids in SEQ ID NO:1 by the integer definingthe percent identity divided by 100 and then subtracting that productfrom said total number of nucleic acids in SEQ ID NO:1, or:n _(n)≦x_(n)−(x _(n) ·y),wherein n_(n) is the number of nucleic acid alterations, x_(n) is thetotal number of nucleic acids in SEQ ID NO:1, y is, for instance 0.70for 70%, 0.80 for 80%, 0.85 for 85% etc., · is the symbol for themultiplication operator, and wherein any non-integer product of x_(n)and y is rounded down to the nearest integer prior to subtracting itfrom x_(n).

(2) Polypeptide embodiments further include an isolated polypeptidecomprising a polypeptide having at least a 50, 60, 70, 80, 85, 90, 95,97 or 100% identity to the polypeptide reference sequence of SEQ IDNO:2, wherein said polypeptide sequence may be identical to thereference sequence of SEQ ID NO:2 or may include up to a certain integernumber of amino acid alterations as compared to the reference sequence,wherein said alterations are selected from the group consisting of atleast one amino acid deletion, substitution, including conservative andnon-conservative substitution, or insertion, and wherein saidalterations may occur at the amino- or carboxy-terminal positions of thereference polypeptide sequence or anywhere between those terminalpositions, interspersed either individually among the amino acids in thereference sequence or in one or more contiguous groups within thereference sequence, and wherein said number of amino acid alterations isdetermined by multiplying the total number of amino acids in SEQ ID NO:2by the integer defining the percent identity divided by 100 and thensubtracting that product from said total number of amino acids in SEQ IDNO:2, or:n _(a) ≦x _(a)−(x _(a) ·Y)wherein n_(a) is the number of amino acid alterations, x_(a) is thetotal number of amino acids in SEQ ID NO:2, y is 0.50 for 50%, 0.60 for60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for95%, 0.97 for 97% or 1.00 for 100%, and · is the symbol for themultiplication operator, and wherein any non-integer product of x_(a)and y is rounded down to the nearest integer prior to subtracting itfrom x_(a).

By way of example, a polypeptide sequence of the present invention maybe identical to the reference sequence of SEQ ID NO:2, that is it may be100% identical, or it may include up to a certain integer number ofamino acid alterations as compared to the reference sequence such thatthe percent identity is less than 100% identity. Such alterations areselected from the group consisting of at least one amino acid deletion,substitution, including conservative and non-conservative substitution,or insertion, and wherein said alterations may occur at the amino- orcarboxy-terminal positions of the reference polypeptide sequence oranywhere between those terminal positions, interspersed eitherindividually among the amino acids in the reference sequence or in oneor more contiguous groups within the reference sequence. The number ofamino acid alterations for a given % identity is determined bymultiplying the total number of amino acids in SEQ ID NO:2 by theinteger defining the percent identity divided by 100 and thensubtracting that product from said total number of amino acids in SEQ IDNO:2, or:n _(a) ≦x _(a)−(x _(a) ·y),wherein n_(a) is the number of amino acid alterations, x_(a) is thetotal number of amino acids in SEQ ID NO:2, y is, for instance 0.70 for70%, 0.80 for 80%, 0.85 for 85% etc., and · is the symbol for themultiplication operator, and wherein any non-integer product of x_(a)and y is rounded down to the nearest integer prior to subtracting itfrom x_(a).

“Individual(s),” when used herein with reference to an organism, means amulticellular eukaryote, including, but not limited to a metazoan, amammal, an ovid, a bovid, a simian, a primate, and a human.

“Isolated” means altered “by the hand of man” from its natural state,i.e., if it occurs in nature, it has been changed or removed from itsoriginal environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not “isolated,”but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated”, as the term is employedherein. Moreover, a polynucleotide or polypeptide that is introducedinto an organism by transformation, genetic manipulation or by any otherrecombinant method is “isolated” even if it is still present in saidorganism, which organism may be living or non-living.

“Polynucleotide(s)” generally refers to any polyribonucleotide orpolydeoxyribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA including single and double-stranded regions.

“Variant” refers to a polynucleotide or polypeptide that differs from areference polynucleotide or polypeptide, but retains essentialproperties. A typical variant of a polynucleotide differs in nucleotidesequence from another, reference polynucleotide. Changes in thenucleotide sequence of the variant may or may not alter the amino acidsequence of a polypeptide encoded by the reference polynucleotide.Nucleotide changes may result in amino acid substitutions, additions,deletions, fusions and truncations in the polypeptide encoded by thereference sequence, as discussed below. A typical variant of apolypeptide differs in amino acid sequence from another, referencepolypeptide. Generally, differences are limited so that the sequences ofthe reference polypeptide and the variant are closely similar overalland, in many regions, identical. A variant and reference polypeptide maydiffer in amino acid sequence by one or more substitutions, additions,deletions in any combination. A substituted or inserted amino acidresidue may or may not be one encoded by the genetic code. A variant ofa polynucleotide or polypeptide may be a naturally occurring such as anallelic variant, or it may be a variant that is not known to occurnaturally. Non-naturally occurring variants of polynucleotides andpolypeptides may be made by mutagenesis techniques or by directsynthesis.

“Disease(s)” means any disease caused by or related to infection by abacteria, including, for example, otitis media in infants and children,pneumonia in elderlies, sinusitis, nosocomial infections and invasivediseases, chronic otitis media with hearing loss, fluid accumulation inthe middle ear, auditive nerve damage, delayed speech learning,infection of the upper respiratory tract and inflammation of the middleear.

EXAMPLES

The examples below are carried out using standard techniques, which arewell known and routine to those of skill in the art, except whereotherwise described in detail. The examples are illustrative, but do notlimit the invention.

Example 1 Cloning of the BASB231 Gene from Non typeable Haemophilusinfluenzae Strain 3224A

Genomic DNA is extracted from the non typeable Haemophilus influenzaestrain 3224A from 10¹⁰ bacterial cells using the QIAGEN genomic DNAextraction kit (Qiagen Gmbh). This material (1 g) is then submitted toPolymerase Chain Reaction DNA amplification using two specific primers.A DNA fragment is obtained, digested by the suitable restrictionendonucleases and inserted into the compatible sites of the pETcloning/expression vector (Novagen) using standard molecular biologytechniques (Molecular Cloning, a Laboratory Manual, Second Edition, Eds:Sambrook, Fritsch & Maniatis, Cold Spring Harbor press 1989).Recombinant pET-BASB231 is then submitted to DNA sequencing using theBig Dyes kit (Applied biosystems) and analyzed on a ABI 373/A DNAsequencer in the conditions described by the supplier.

Example 2 Expression and Purification of Recombinant BASB231 Protein inEscherichia coli

The construction of the pET-BASB231 cloning/expression vector isdescribed in Example 1. This vector harbours the BASB231 gene isolatedfrom the non typeable Haemophilus influenzae strain 3224A in fusion witha stretch of 6 Histidine residues, placed under the control of thestrong bacteriophage T7 gene 10 promoter. For expression study, thisvector is introduced into the Escherichia coli strain Novablue (DE3)(Novagen), in which, the gene for the T7 polymerase is placed under thecontrol of the isopropyl-beta-D thiogalactoside (IPTG)-regulatable lacpromoter. Liquid cultures (100 ml) of the Novablue (DE3) [pET-BASB231]E. coli recombinant strain are grown at 37° C. under agitation until theoptical density at 600 nm (OD600) reached 0.6. At that time-point, IPTGis added at a final concentration of 1 mM and the culture is grown for 4additional hours. The culture is then centrifuged at 10,000 rpm and thepellet is frozen at −20° C. for at least 10 hours. After thawing, thepellet is resuspended during 30 min at 25° C. in buffer A (6M guanidinehydrochloride, 0.1M NaH2PO4, 0.01M Tris, pH 8.0), passed three-timesthrough a needle and clarified by centrifugation (20000 rpm, 15 min).The sample is then loaded at a flow-rate of 1 ml/min on a Ni2+-loadedHitrap column (Pharmacia Biotech). After passsage of the flowthrough,the column is washed succesively with 40 ml of buffer B (8M Urea,0.1MNaH2PO4, 0.01M Tris, pH 8.0), 40 ml of buffer C (8M Urea,0.1MNaH2PO4, 0.01M Tris, pH 6.3). The recombinant protein BASB231/His6is then eluted from the column with 30 ml of buffer D (8M Urea,0.1MNaH2PO4, 0.01M Tris, pH 6.3) containing 500 mM of imidazole and 3ml-size fractions are collected. Highly enriched BASB231/His6 proteincan be eluted from the column. This polypeptide is detected by a mousemonoclonal antibody raised against the 5-histidine motif. Moreover, thedenatured, recombinant BASB231-His6 protein is solubilized in a solutiondevoid of urea. For this purpose, denatured BASB231-His6 contained in 8Murea is extensively dialyzed (2 hours) against buffer R (NaCl 150 mM, 10mM NaH2PO4, Arginine 0.5M pH6.8) containing successively 6M, 4M, 2M andno urea. Alternatively, this polypeptide is purified undernon-denaturing conditions using protocoles described in theQuiexpresssionist booklet (Qiagen Gmbh).

Example 3 Production of Antisera to Recombinant BASB231

Polyvalent antisera directed against the BASB231 protein are generatedby vaccinating rabbits with the purified recombinant BASB231 protein.Polyvalent antisera directed against the BASB231 protein are alsogenerated by vaccinating mice with the purified recombinant BASB231protein. Animals are bled prior to the first immunization (“pre-bleed”)and after the last immunization.

Anti-BASB231 protein titers are measured by an ELISA using purifiedrecombinant BASB231 protein as the coating antigen. The titer is definedas mid-point titers calculated by 4-parameter logistic model using theXL Fit software. The antisera are also used as the first antibody toidentify the protein in a western blot as described in example 5 below.

Example 4 Immunological Characterization: Surface Exposure of BASB231

Anti-BASB231 protein titres are determined by an ELISA usingformalin-killed whole cells of non typable Haemophilus influenzae(NTHi). The titer is defined as mid-point titers calculated by4-parameter logistic model using the XL Fit software.

Example 5 Immunological Characterisation: Western Blot Analysis

Several strains of NTHi, as well as clinical isolates, are grown onChocolate agar plates for 24 hours at 36° C. and 5% CO₂. Severalcolonies are used to inoculate Brain Heart Infusion (BHI) brothsupplemented by NAD and hemin, each at 10 μg/ml. Cultures are grownuntil the absorbance at 620 nm is approximately 0.4 and cells arecollected by centrifugation. Cells are then concentrated and solubilizedin PAGE sample buffer. The solubilized cells are then resolved on 4-20%polyacrylamide gels and the separated proteins are electrophoreticallytransferred to PVDF membranes. The PVDF membranes are then pretreatedwith saturation buffer. All subsequent incubations are carried out usingthis pretreatment buffer.

PVDF membranes are incubated with preimmune serum or rabbit or mouseimmune serum. PVDF membranes are then washed.

PVDF membranes are incubated with biotin-labeled sheep anti-rabbit ormouse Ig. PVDF membranes are then washed 3 times with wash buffer, andincubated with streptavidin-peroxydase. PVDF membranes are then washed 3times with wash buffer and developed with 4chloro-1-naphtol.

Example 6 Immunological Characterization: Bactericidal Activity

Complement-mediated cytotoxic activity of anti-BASB231 antibodies isexamined to determine the vaccine potential of BASB231 protein antiserumthat is prepared as described above. The activities of the pre-immuneserum and the anti-BASB231 antiserum in mediating complement killing ofNTHi are examined.

Strains of NTHi are grown on plates. Several colonies are added toliquid medium. Cultures are grown and collected until the A620 isapproximately 0.4. After one wash step, the pellet is suspended anddiluted.

Preimmune sera and the anti-BASB231 sera are deposited into the firstwell of a 96-wells plate and serial dilutions are deposited in the otherwells of the same line. Live diluted NTHi is subsequently added and themixture is incubated. Complement is added into each well at a workingdilution defined beforehand in a toxicity assay.

Each test includes a complement control (wells without serum containingactive or inactivated complement source), a positive control (wellscontaining serum with a know titer of bactericidal antibodies), aculture control (wells without serum and complement) and a serum control(wells without complement).

Bactericidal activity of rabbit or mice antiserum (50% killing ofhomologous strain) is measured.

Example 7 Presence of Antibody to BASB231 in Human Convalescent Sera

Western blot analysis of purified recombinant BASB231 is performed asdescribed in Example 5 above, except that a pool of human sera fromchildren infected by NTHi is used as the first antibody preparation.

Example 8 Efficacy of BASB231 Vaccine: Enhancement of Lung Clearance ofNTHi in Mice

This mouse model is based on the analysis of the lung invasion by NTHifollowing a standard intranasal challenge to vaccinated mice.

Groups of mice are immunized with BASB231 vaccine. After the booster,the mice are challenged by instillation of bacterial suspension into thenostril under anaesthesia.

Mice are killed between 30 minutes and 24 hours after challenge and thelungs are removed aseptically and homogenized individually. The log10weighted mean number of CFU/lung is determined by counting the coloniesgrown on agar plates after plating of dilutions of the homogenate. Thearithmetic mean of the log10 weighted mean number of CFU/lung and thestandard deviations are calculated for each group.

Results are analysed statistically.

In this experiment groups of mice are immunized either with BASB231 orwith a killed whole cells (kwc) preparation of NTHi or sham immunized.

Example 9 Inhibition of NTHi Adhesion onto Cells by Anti-BASB231Antiserum

This assay measures the capacity of anti BASB231 sera to inhibit theadhesion of NTHi bacteria to epithelial cells. This activity couldprevent colonization of the nasopharynx by NTHi.

One volume of bacteria is incubated on ice with one volume of pre-immuneor anti-BASB231 immune serum dilution. This mixture is subsequentlyadded in the wells of a 24 well plate containing a confluent cellsculture that is washed once with culture medium to remove traces ofantibiotic. The plate is centrifuged and incubated.

Each well is then gently washed. After the last wash, sodiumglycocholate is added to the wells. After incubation, the cell layer isscraped and homogenised. Dilutions of the homogenate are plated on agarplates and incubated. The number of colonies on each plate is countedand the number of bacteria present in each well calculated.

Deposited Materials

A deposit of strain 3 (strain 3224A) has been deposited with theAmerican Type Culture Collection (ATCC) on May 5, 2000 and assigneddeposit number PTA-1816.

The non typeable Haemophilus influenza strain deposit is referred toherein as “the deposited strain” or as “the DNA of the depositedstrain.”

The deposited strain contains a full length BASB231 polynucleotidesequence.

The sequence of the polynucleotides contained in the deposited strain,as well as the amino acid sequence of any polypeptide encoded thereby,are controlling in the event of any conflict with any description ofsequences herein.

The deposit of the deposited strain has been made under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicro-organisms for Purposes of Patent Procedure. The deposited strainwill be irrevocably and without restriction or condition released to thepublic upon the issuance of a patent. The deposited strain is providedmerely as convenience to those of skill in the art and is not anadmission that a deposit is required for enablement, such as thatrequired under 35 U.S.C. §112. A license may be required to make, use orsell the deposited strain, and compounds derived therefrom, and no suchlicense is hereby granted.

1-30. (canceled)
 31. An isolated polypeptide comprising a memberselected from the group consisting of: (a) an amino acid sequence whichhas at least 85% identity to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,56, 58, 60, 62, 64, 66, 68, 70 or 72 over the entire length of saidsequence; and (b) an immunogenic fragment of SEQ ID NO: 2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70 or 72, wherein theimmunogenic fragment has substantially the same immunogenic activity asSEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68,70 or
 72. 32. The isolated polypeptide of claim 31, wherein the aminoacid sequence of (a) has at least 95% identity to SEQ ID NO: 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70 or 72 over the entirelength of said sequence.
 33. The isolated polypeptide of claim 31,comprising SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,64, 66, 68, 70 or
 72. 34. The isolated polypeptide of claim 31,consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,64, 66, 68, 70 or
 72. 35. The isolated polypeptide of claim 31, whereinthe isolated polypeptide is an immunogenic fragment of SEQ ID NO: 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70 or 72,wherein the immunogenic fragment has substantially the same immunogenicactivity as SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,64, 66, 68, 70 or
 72. 36. The isolated polypeptide of claim 31, whereinthe polypeptide is part of a larger fusion protein.
 37. An isolatedpolynucleotide encoding a polypeptide of claim
 31. 38. The isolatedpolynucleotide of claim 37, wherein the isolated polynucleotidecomprises a nucleotide sequence that encodes a polypeptide selected fromSEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68,70 or
 72. 39. An isolated polynucleotide comprising a nucleotidesequence that has at least 85% identity to SEQ ID NO: 1, 3, 5, 7, 9, 11,13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47,49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71 or 73; or the fullcomplement to said isolated polynucleotide.
 40. The isolatedpolynucleotide of claim 39, wherein the nucleotide sequence has at least95% identity to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59,61, 63, 65, 67, 69, 71 or
 73. 41. The isolated polynucleotide of claim39, wherein the isolated polynucleotide comprises SEQ ID NO: 1, 3, 5, 7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43,45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71 or
 73. 42. Theisolated polynucleotide of claim 39, wherein the isolated polynucleotideconsists of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61,63, 65, 67, 69, 71 or
 73. 43. An isolated polynucleotide, comprising anucleotide sequence encoding a polypeptide selected from SEQ ID NO: 2,4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70 or 72obtainable by screening an appropriate library under stringenthybridization conditions with a labeled probe having the correspondingDNA sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59,61, 63, 65, 67, 69, 71 or 73, or a fragment thereof.
 44. An expressionvector or a recombinant live microorganism comprising an isolatedpolynucleotide according to claim
 37. 45, A host cell comprising theexpression vector or a subcellular fraction or a membrane of said hostcell expressing an isolated polypeptide of claim
 31. 46. A process forproducing the polypeptide expressed by the host cell of claim 45,comprising culturing the host cell under conditions sufficient for theproduction of said polypeptide and recovering the polypeptide from theculture medium.
 47. A process for expressing a polynucleotide of claim37, comprising transforming a host cell with the expression vectorcomprising said polynucleotide and culturing said host cell underconditions sufficient for expression of said polynucleotide.
 48. Animmunogenic composition comprising an effective amount of the isolatedpolypeptide of claim 31, and a pharmaceutically effective carrier. 49.The immunogenic composition according to claim 48, wherein saidimmunogenic composition comprises at least one other non typeable H.influenzae antigen.
 50. An immunogenic composition comprising aneffective amount of the polynucleotide of claim
 37. 51. An antibodyimmunospecific for a polypeptide comprising a member selected from: a)an amino acid sequence which has at least 85% identity to SEQ ID NO: 2,4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70 or 72 overthe entire length of said sequence; and b) an immunogenic fragment ofSEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68,70 or 72, wherein the immunogenic fragment has substantially the sameimmunogenic activity as SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,58, 60, 62, 64, 66, 68, 70 or
 72. 52. A method of diagnosing a nontypeable H. influenzae infection, comprising identifying a polypeptidecomprising a member selected from: a) an amino acid sequence which hasat least 85% identity to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,58, 60, 62, 64, 66, 68, 70 or 72 over the entire length of saidsequence; and b) an immunogenic fragment of SEQ ID NO: 2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70 or 72, wherein theimmunogenic fragment has substantially the same immunogenic activity asSEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68,70 or 72; or an antibody that is immunospecific for said polypeptide,present within a biological sample from an animal suspected of havingsuch an infection.
 53. A method of diagnosing a non typeable H.influenzae infection or the presence of non typeable H. influenzae in asample, comprising the step of identifying the stringent hybridisationof a polynucleotide probe comprising at least 15 nucleotides from apolynucleotide selected from SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53,55, 57, 59, 61, 63, 65, 67, 69, 71 or 73 to bacterial genomic DNApresent within a sample, optionally a biological sample taken from ananimal suspected of having a non typeable H. influenzae infection.
 54. Atherapeutic composition useful in treating humans with non typeable H.influenzae disease comprising at least one antibody directed against apolypeptide selected from: a) an amino acid sequence which has at least85% identity to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70 or 72 over the entire length of said sequence; b) animmunogenic fragment of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,58, 60, 62, 64, 66, 68, 70 or 72, wherein the immunogenic fragment hassubstantially the same immunogenic activity as SEQ ID NO: 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70 or 72; and, asuitable pharmaceutical carrier.
 55. A method of generating an immuneresponse in an animal comprising administering an immunogeniccomposition comprising an immunologically effective amount of apolypeptide selected from: a) an amino acid sequence which has at least85% identity to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70 or 72 over the entire length of said sequence; (b) animmunogenic fragment of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,58, 60, 62, 64, 66, 68, 70 or 72, wherein the immunogenic fragment hassubstantially the same immunogenic activity as SEQ ID NO: 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70 or 72; to the animal.56. A method of generating an immune response in an animal, comprisingadministering an immunogenic composition comprising an immunologicallyeffective amount of a polynucleotide that has at least 85% identity toSEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67,69, 71 or
 73. 57. A mutated ntHi strain, wherein the gene shown in SEQID NO:1 has been engineered such that it either expresses its geneproduct constitutively, or it has been substantially knocked-out so asto switch off functional expression of its gene product. 58.Lipo-oligosaccharide isolated from the mutated ntHi strain of claim 57.59. A method for preparing an oligosaccharide in vitro comprising thesteps of contacting a reaction mixture comprising an activatedsaccharide residue to an acceptor moiety comprising a further saccharideresidue in the presence of the glycosyltransferase having an amino acidsequence of SEQ ID NO:2, or a functionally active fragment thereof.